Current and emerging therapies for fungal biofilms and systemic infections

Fungal biofilm infections remain a complex and urgent challenge as current antifungals are limited by toxicity, suboptimal efficacy, and their potential to develop antifungal resistance. While existing antifungals are still the cornerstone of treatment, their reduced efficacy against biofilms highlights the importance of elucidating effective therapeutic methods and novel compounds against these biofilm infections. The discovery and development of novel antifungals have shown significant potential to address rising resistance and improve treatment of fungal systemic and biofilm infections. Repurposed drugs provide a cost-effective, low-risk path to clinical use, while natural compounds may serve as complementary agents to prevent and inhibit biofilm formation. Beyond pharmacological approaches, emerging technologies such as photodynamic therapy, surface coatings, and nanotechnology-based drug delivery systems present exciting opportunities to prevent and eradicate fungal biofilms at their source. Importantly, many of these innovations have demonstrated broad-spectrum activity against medically important fungi including opportunistic C. albicans, C. neoformans , and A. fumigatus . Continued investment in fundamental research and translational studies is essential to fully elucidate the potential of emerging antifungal therapies to be applied for broader clinical use. Achieving this goal will require a collaborative effort among academic institutions and industry partners to bridge these new therapies to clinical practice. The future of fungal biofilm treatment rests on the successful integration of novel agents and innovative technologies with a patient-centered focus, which prioritizes efficacy and safety, and prevents resistance development. Ultimately, these next-generation therapies have the potential to transform clinical outcomes by disrupting and preventing fungal biofilms while also keeping the patient’s safety a top priority.

Fungal pathogens are responsible for more than 1 billion infections annually, resulting in an estimated 1.6 million deaths worldwide ( Rokas 2022 ). Fungal diseases vary from superficial infections to life-threatening invasive fungal diseases, especially in immunocompromised patients. The World Health Organization classified fungal pathogens, including Cryptococcus neoformans , Candida albicans , and Aspergillus fumigatus , as critical priority, aiming to steer research, drug development, and public health strategies to counter the escalating threat of fungal infections ( Parums 2022 ). Fungal biofilms represent one of the most significant and prevalent concerns in medicine, due to the innate barrier they possess against effective treatments ( Martinez and Fries 2010 ). Biofilms are made up of fungal cells that attach to a surface and create a self-enclosed community with an extracellular matrix (ECM), which consists of polysaccharides such as β-glucans, and mannans ( Robertson and Casadevall 2009 ; Zarnowski et al. 2014 ; Mitchell et al. 2015 ; Mitchell et al. 2016 ). These characteristics make biofilm-derived cells more than 1000-fold resistant to antifungals, compared to planktonic cells ( Ramage et al. 2012 ). Additionally, biofilms commonly develop on abiotic surfaces such as catheters, prosthetic joints, heart valves, and pacemakers, as well as on biotic surfaces, such as living tissues within the host, posing a substantial risk of healthcare-associated infections ( Figure 1 ) ( Martinez and Fries 2010 ). These infections lead to worse prognosis, longer hospital stays, and higher medical costs than their planktonic counterparts, hence the importance of elucidating the dire need for effective treatments. Fungal biofilms display numerous resistance mechanisms. ECM acts as a protective barrier, making it more challenging for traditional antifungals to penetrate this barrier and exert their effect on the biofilm-derived cells ( Figure 2 ) ( Mukherjee et al. 2003 ; Taff et al. 2013 ; Mitchell et al. 2016 ; Mannan et al. 2024 ). Biofilms also harbor persister cells, metabolically quiescent subpopulations that are capable of surviving high antifungal concentrations and resisting environmental stressors and host immune defenses ( Mitchell et al. 2016 ; Kaur and Nobile 2023 ). However, the existence of persister cells in C. albicans biofilms remains debated, with some studies failing to detect them ( Denega et al. 2019 ). Beyond structural defenses, biofilms employ active resistance mechanisms, such as upregulated efflux pumps ( Ramage et al. 2012 ). During the early biofilm stages of C. albicans , efflux pumps are upregulated, which results in actively exporting azole antifungals out of the fungal cell, thereby reducing intracellular drug accumulation and diminishing therapeutic efficacy ( Taff et al. 2013 ). The most well-characterized efflux pumps in fungal pathogens include the ATP-binding cassette (ABC) transporters encoded by CDR1 and CDR2 genes, and the major facilitator superfamily (MFS) transporter encoded by MDR1 ( Todd 1975 ). Their expression is significantly upregulated upon exposure to azoles, a response often mediated by gain-of-function mutations in specific transcriptional regulators, such as TAC1 for CDR1 and CDR2 , and MRR1 for MDR1 ( Morio et al. 2013 ). These mutations result in constitutive overexpression of efflux pumps, thereby conferring stable resistance phenotypes in clinical isolates. However, this overexpression tends to diminish in mature biofilm ( Taff et al. 2013 ; Tsui et al. 2016 ). Other biofilm resistance mechanisms include the alteration of their cellular metabolic states ( Liu et al. 2022 ) and activation of stress responses ( Iyer et al. 2022 ). These biofilm-specific mechanisms create a particularly formidable challenge to treatment, complicating the management of biofilm-associated infections and severely limiting available therapeutic options ( Ramage et al. 2012 ; Pierce et al. 2013 ). In response to the challenge of resistant fungal biofilms, ongoing efforts focus on novel approaches and treatment options that target biofilm-specific structural features such as their ECM ( Roy and Gow 2023 ). Simply modifying current treatment options may not be as effective, due to increased resistance and host toxicity at higher drug doses ( Andes et al. 2009 ). As a result, this review provides a comprehensive analysis of current and emerging treatment options against medically important fungal pathogens, including C. albicans, C. neoformans , and A. fumigatus as they make up three of the most common fungal pathogens with well-characterized biofilm structures ( Fanning and Mitchell 2012 ). It highlights the progress on elucidating the efficacy of novel antifungal options, drug repurposing, natural products, and other therapeutic strategies, aiming to improve patient outcomes and combat the growing challenge of biofilm-related antifungal resistance. C. albicans is a commensal fungus commonly found on the human mucosal surfaces ( Neville et al. 2015 ). However, in immunocompromised individuals, it overgrows and transitions into life-threatening opportunistic pathogen, causing invasive candidiasis ( Valand and Girija 2021 ). It is a leading cause of nosocomial infections, responsible for 15% of sepsis cases, and 40% of bloodstream infections ( Gulati and Nobile 2016 ). Its virulence is linked to its ability to undergo morphological switching between a yeast and hyphae, with the hyphal form containing more chitin, providing additional structural protection against extracellular conditions ( Talapko et al. 2021 ). Biofilm formation is driven by cell wall adhesins that facilitate attachment to host cells ( Nobile et al. 2008 ). During biofilm development, the filamentous form predominates, as this morphology supports a robust three-dimensional structure and facilitates ECM production ( Desai and Mitchell 2015 ). A key component of this matrix is β-1,3-glucan, which sequesters antifungals within the ECM, thereby limiting their penetration and access to cellular targets ( Nett et al. 2007 ). Additionally, β-1,3 glucan impairs host immunity by suppressing neutrophil activation, further promoting the fungal biofilm resistance to innate immune system ( Mitchell et al. 2016 ). These biofilm properties adhere strongly to both tissue and abiotic surfaces used in medical procedures. Given the increasing prevalence of biofilm-associated infections and the emergence of non- albicans Candida species in hospital environments, it is critical to evaluate the current treatment landscape to combat biofilm infections. C. auris , in particular, has emerged as a multidrug-resistant responsible for hospital outbreaks worldwide ( Sabino et al. 2020 ). Alarmingly, some clinical isolates of C. auris has been reported as pan-resistant, rendering all three classes of antifungals ineffective ( O’Brien et al. 2020 ), with persistent biofilms on medical devices and hospital surfaces, further contributing to its antifungal resistance ( Horton and Nett 2020 ; Elgammal et al. 2024 ). Thus, there is an urgent need to develop novel antifungals and expand the current antifungal armamentarium to effectively manage these challenging infections. While the three major classes of antifungals (azoles, polyenes, and echinocandins) are widely used to treat planktonic infections, their efficacy is markedly reduced in treating biofilm infections ( Costa-de-Oliveira and Rodrigues 2020 ). Polyenes, mainly amphotericin B, exhibit potent broad-spectrum fungicidal activity by binding to ergosterol and forming pores in their target cells. However, under biofilm conditions, amphotericin B has been shown to be up to 10-fold less effective, compared to its activity against planktonic cells ( Ajetunmobi et al. 2023 ). One option is to increase the antifungal concentration used during infection to increase their fungicidal effect. However, this approach is clinically impractical due to drug toxicity, especially nephrotoxicity when applied to polyenes ( Fisher et al. 2022 ). Azoles, which target the ergosterol biosynthesis pathway by targeting lanosterol 14α-demethylase, offer a less toxic alternative to polyenes. However, azole resistance in C. albicans has become more prevalent throughout the years, particularly within biofilms ( Gao et al. 2018 ). Echinocandins inhibit β-glucan synthesis in the fungal cell wall, making them more effective than the other two classes at targeting biofilms ( Sucher et al. 2009 ). Their fungicidal activity and favorable safety profile have positioned echinocandins as the front-line agents for the treatment of candidiasis. Nonetheless, the emergence of mutations of the FKS genes, which encode for the echinocandins target enzyme, results in reducing their efficacy ( Ajetunmobi et al. 2023 ). While the most common intervention for managing C. albicans biofilm infections involves the removal of the infected implants, it poses high risk for patients undergoing surgery, including surgical complications and substantial healthcare costs, exceeding $50 000 per patient ( Martinez and Fries 2010 ). These limitations clearly highlight the urgent need for future therapeutic development to combat C. albicans biofilms. C. neoformans is one of the leading causes of meningoencephalitis in immunocompromised individuals ( Rodrigues et al. 1999 ). Its virulence is mainly attributed to its polysaccharide capsule, which is primarily composed of glucuronoxylomannan (GXM). Secretion of GXM not only affects the host immune response but is also necessary for biofilm formation, as it contributes significantly to the establishment of ECM ( Martinez and Casadevall 2005 ). C. neoformans has been shown to form brain cryptococcomas, biofilm-like structures ( Aslanyan et al. 2017 ), making its biofilm-forming capacity clinically relevant. Moreover, the resistance mechanisms have made current treatment options more futile and left patients with these persistent brain biofilm-like infections with little options, highlighting the need for further elucidation of treatment options for C. neoformans biofilms. A combination of liposomal amphotericin B and 5-flurocytosine, followed by fluconazole, has been the recommended treatment regimen for pulmonary cryptococcosis and cryptococcal meningitis. This treatment regimen remains effective against planktonic C. neoformans but shows reduced efficacy against fungal biofilm ( Martinez and Casadevall 2006 ; Sionov et al. 2013 ; Bongomin et al. 2018 ). Resistance mechanisms of C. neoformans to fluconazole include mutations in the ERG11 gene, encoding 14α-demethylase, the primary target of azoles, leading to decreased drug susceptibility. These challenges underscore the urgent need for alternative therapies capable of effectively targeting C. neoformans biofilms. A. fumigatus is an opportunistic fungal pathogen and the primary cause of invasive aspergillosis, particularly to immunocompromised individuals ( Kwon-Chung and Sugui 2013 ). Its pathogenesis is intricately tied to its ability to form dense, interwoven hyphae-based biofilms, which follow a multi-phase developmental process ( Figure 3 ) ( Morelli et al. 2021 ). Additionally, the pathogen’s ubiquity is attributed to its adaptability to various environmental conditions, effective air dispersal, and ability to reach distal airways ( Kwon-Chung and Sugui 2013 ). A. fumigatus can also form aspergilloma, a localized infection consisting of hyphae and fungal mass, which contributes to biofilm-associated infections, and can be detected through bronchopulmonary lavage in patients with aspergillosis ( Ramage et al. 2012 ). Current treatments for A. fumigatus infections primarily rely on mold-active triazoles, such as voriconazole. itraconazole, posaconazole, and isavuconazole, as well as amphotericin B ( Rybak et al. 2019 ). These agents are effective against most susceptible strains; however, the global emergence of triazole-resistant A. fumigatus strains has significantly compromised treatment efficacy, posing a major public health concern ( Wiederhold and Verweij 2020 ). Resistance mechanisms include point mutations in the cyp51A gene, which alter the target site of azoles, as well as overexpression of efflux pumps that reduce intracellular drug concentrations ( Gonçalves et al. 2020 ). When azole-resistant A. fumigatus is suspected or confirmed, amphotericin B is often employed as first-line therapy due to its broad-spectrum activity, despite its potential nephrotoxicity ( Novak et al. 2020 ; Arastehfar et al. 2021 ). Liposomal formulations of amphotericin B, which are less toxic than the conventional form, are increasingly utilized to mitigate these adverse effects ( Tollemar et al. 2001 ). Combination therapies have shown promise as salvage treatments against azole-resistant strains. For example, echinocandins in combination with posaconazole or isavuconazole may offer synergistic effects in resistant isolates, although clinical evidence remains limited ( Tollemar et al. 2001 ; Novak et al. 2020 ; Arastehfar et al. 2021 ). Unlike azoles, and polyenes, echinocandins act exclusively on the cell wall, making them highly specific and reducing cross-resistance with other antifungal classes ( Patterson et al. 2016 ). Nonetheless, resistance and limited therapeutic options pose significant clinical challenges. Addressing this requires enhanced antifungal stewardship, routine resistance surveillance, and the development of new antifungal agents or safer formulations of existing drugs. The development of novel antifungal agents is essential for addressing systemic and biofilm-associated fungal infections. Several emerging therapies are designed to penetrate or disrupt biofilms, thereby enhancing treatment efficacy and overcoming antifungal resistance. In this context, we aim to delve into the therapeutic potential of these promising agents in treating clinically significant fungal biofilm and systemic infections. Due to its high prevalence in nosocomial infections, C. albicans has many promising candidates for potential as antifungals targeting biofilm ( Figure 4 ). Rezafungin, an FDA- approved second generation of echinocandins with improved pharmacokinetics. It offers once-weekly dosing due to its long half-life (130 h) instead of daily administration required for other echinocandins ( Lepak et al. 2018 ). Additionally, rezafungin disrupts biofilm formation, decreases the mature biofilms’ metabolic activity and thickness, and inhibits hyphae formation, showcasing its superior stability compared to fluconazole ( Chandra and Ghannoum 2018 ). Ibrexafungerp, a semisynthetic triterpenoid derived from enfumafungin, is the first orally bioavailable glucan synthase inhibitor with high tissue distribution ( Angulo et al. 2022 ). It has demonstrated broad-spectrum activity against many Candida species, including C. albicans , and has received the FDA approval for the treatment of vulvovaginal candidiasis ( Phillips et al. 2023 ). Although it shares the same mechanism of action with echinocandins, the partially overlapping binding sites of ibrexafungerp and echinocandins result in distinct hot-spot mutations and limit cross-resistance ( Lamoth et al. 2022 ). Furthermore, ibrexafungerp has demonstrated in vitro superiority over fluconazole, reducing Candida biofilm metabolic activity by 50% at a concentration of 0.25 μg/mL, compared to >256 μg/mL for fluconazole ( Gamal et al. 2021 ). Fosmanogepix is a novel antifungal prodrug that is converted into its active form, manogepix, by alkaline phosphatases ( Almajid et al. 2024 ). Currently in Phase III for the treatment of candidemia and invasive candidiasis, fosmanogepix disrupts the fungal cell wall by inhibiting inositol acetyltransferase Gwt1, which plays a role in the glycosylphosphatidylinositol biosynthesis pathway. Additionally, manogepix impairs several virulence factors in C. albicans , such as adhesion to surfaces, hyphal morphogenesis, and the formation of biofilms ( Shaw and Ibrahim 2020 ). Furthermore, its excellent oral bioavailability (>90%) and broad-spectrum antifungal activity underscore its potential as a promising therapeutic option for invasive fungal infection ( Phillips et al. 2023 ). Turbinmicin, isolated from the sea squirt microbiome, has demonstrated potent activity against C. albicans biofilms by targeting Sec14 , which plays a role in the vesicle dependent delivery of the biofilm matrix ( Zhao et al. 2021 ). Notably, turbinmicin significantly decreased the vesicle delivery of the matrix, eradicated the biofilm matrix, and reduced the metabolic activity of biofilms at extremely low concentrations ( Zhao et al. 2021 ). Lastly, the in vivo biofilm efficacy of turbinmicin was evaluated in a rat catheter model. At 2.5 μg/mL, turbinmicin significantly reduced biofilm-derived CFU by ~2 log 10 , and it completely sterilized the infected catheters at 10 μg/mL ( Zhao et al. 2021 ). Given the risk of device-associated fungal infections, turbinmicin’s ability to sterilize catheters emphasizes its potential as a less invasive and more effective treatment option. Tetrazoles, such as VT-1161 (oteseconazole) and VT-1598, represent the fourth generation of azole antifungals with improved specificity (2000-fold) for fungal CYP51, and reduced mammalian toxicity ( Break et al. 2018 ; Maione et al. 2023 ; Ordaya et al. 2023 ). VT-1161, an FDA-approved drug for the treatment of recurrent vulvovaginal candidiasis, inhibits C. albicans biofilm by 85% at 0.5 μg/mL. Additionally VT-1598 demonstrated efficacy in murine models of C. auris , C. albicans , and cryptococcosis, showing potential for broad clinical use ( Garvey et al. 2018 ; Wiederhold et al. 2019 ; Garvey et al. 2020 ). The emergence of multidrug-resistant C. albicans strains, particularly those associated with biofilm formation, presents a significant challenge in clinical settings. Thus, the development of novel antifungal agents offers strong alternatives to existing therapies. These agents not only exhibit potent antifungal activity but also target biofilm-specific mechanisms, addressing one of the major limitations of current antifungal treatments. Continued research and clinical trials are essential to validate the efficacy and safety of these candidates, with the goal of enhancing treatment outcomes for patients suffering from invasive C. albicans infections. Although C. neoformans has shown relatively low antifungal resistance, emerging resistance, particularly to fluconazole, is a growing concern, with 10–30% of clinical isolates exhibiting reduced susceptibility and resistance ( Bongomin et al. 2018 ; Zafar et al. 2019 ; Huang et al. 2024 ). Additionally, some clinical isolates of C. neoformans have shown to be resistant to flucytosine, indicating one of the reasons why it is used in combination with amphotericin B ( Merry and Boulware 2016 ). Flucytosine resistance arises primarily from mutations in fcy1 , fcy2 , and fur1 genes, which encode cytosine deaminase, cytosine permease, and uracil phosphoribosyltransferase, respectively. These mutations hinder drug uptake or metabolism within the fungal cell, preventing the conversion of 5-flurocytosine into its active toxic form, 5-fluorouracil, and rendering the antifungal treatment ineffective ( Billmyre et al. 2020 ; Chang et al. 2021 ). In contrast, resistance to amphotericin B is less prevalent than to that of flucytosine. It is important to note that these measures of resistance are against planktonic C. neoformans infections, which do not consider the innate resistance that biofilms provide against antifungals. Additionally, the intrinsic resistance of the fungus to azoles underscores the need to identify alternative therapies that specifically target biofilm-associated infections. Developing such targeted treatments could also reduce the reliance on the standard amphotericin B and flucytosine regimen, thereby mitigating the risk of resistance in C. neoformans. Alongside its effect against C. albicans , fosmanogepix has also been shown to have antifungal activity against C. neoformans by disrupting the fungal cell wall. Derivatives of the Gw1t inhibitor show strong antifungal activity with MIC’s as low as 0.004 μg/mL ( Trzoss et al. 2019 ). In murine models of cryptococcal meningitis, fosmanogepix significantly reduced fungal burden in lung and brain tissues, particularly when used in combination with fluconazole, suggesting potential for combination therapy to enhance efficacy or overcome resistance in clinical settings ( Hoda et al. 2020 ). Given its ability to inhibit C. albicans biofilms, further investigation is warranted into its efficacy against C. neoformans biofilms. The rise of resistance in C. neoformans infections regimens is indicative of the need of novel antifungals to target these challenging infections, in particular those associated with biofilms. Cryptococcomas, with their dense and polysaccharide rich matrix, poses major therapeutic challenges, especially in brain, where the blood-brain barrier further limits drug penetration. This requires prolonged therapy and is often associated with relapse despite extended courses of antifungal therapy ( Perfect et al. 2010 ). Emerging therapies, such as fosmanogepix, represent highly promising therapeutic directions. Moving forward, robust in vivo studies and rigorous clinical investigations will be essential to fully characterize these novel antifungals and successfully translate these advancements into effective treatments for cryptococcal infections. Despite significant advances in antifungal therapy, A. fumigatus remains an alarming pathogen, particularly in immunocompromised populations. Resistance to existing antifungal agents, as well as the complexity of fungal biofilms, emphasizes the need for new treatment options. Several novel anti- Aspergillus therapeutics are in various stages of clinical development, each offering distinct mechanisms of action and formulations that may broaden the therapeutic landscape. Olorofim (F901318), a Phase III clinical drug, offers potent efficacy against difficult-to-treat fungal infections, including those caused by A. fumigatus ( Seyedmousavi et al. 2019 ; Vanbiervliet et al. 2024 ). It inhibits early biofilm formation (~66.4% reduction at 4 h), but it is less effective against mature biofilms ( Kirchhoff et al. 2022 ). This highlights the clinical relevance of early intervention, given the notorious resistance of mature fungal biofilms to treatments. Olorofim targets fungal dihydroorotate dehydrogenase (DHODH), blocking pyrimidine biosynthesis via selective binding to the N-terminal helical domain, with minimal off-target effects due to differences from the human enzyme ( Oliver et al. 2016 ; Kirchhoff et al. 2022 ; Vanbiervliet et al. 2024 ). It retains potency against azole- and amphotericin B-resistant strains and has not shown intrinsic resistance in 975 clinical isolates. However, experimental exposure has revealed resistance potential via pyrE mutations ( Escribano et al. 2022 ; Buil et al. 2022 ). Additionally, olorofim is orally bioavailable with long half-life of 20–30 h, crosses the blood-brain barrier, and distributes well into lungs, liver, and kidneys ( Oliver et al. 2016 ; Kirchhoff et al. 2022 ; Vanbiervliet et al. 2024 ). Its oral dosing is once daily, and clinical trials reported its favorable tolerability with mild gastrointestinal side effects ( Vanbiervliet et al. 2024 ). A notable concern is the antagonistic interaction reported between olorofim and azole antifungals, likely due to azole-induced upregulation of pyrimidine biosynthesis ( van Rhijn et al. 2022 ). Furthermore, exposure of A. fumigatus to ipflufenoquin, an agricultural fungicide with a similar mechanism of action, has been shown to accelerate resistance development to olorofim, potentially undermining its clinical efficacy against invasive aspergillosis ( van Rhijn et al. 2024 ). Opelconazole (PC945), a triazole antifungal, was developed specifically for inhaled administration targeting pulmonary A. fumigatus infections ( Colley et al. 2017 ). Its topical delivery limits systemic exposure and drug interactions common oral triazoles. Owing to its lipophilic properties, it accumulates in the lungs even after treatment is stopped, likely due to sequestration by alveolar macrophages and other pulmonary cells, providing prolonged local activity ( Cass et al. 2021 ). This lung-specific persistence, low systemic distribution, and its robust antifungal activity make PC945 a compelling prophylactic option for high-risk populations, such as lung transplant recipients, ( Eschenauer 2024 ). However, evidence regarding its activity against A. fumigatus biofilms remains limited and warrants further investigation. In addition to its activity against C. albicans , Ibrexafungerp exhibits broad-spectrum activity against Aspergillus species. In experimental models of invasive pulmonary aspergillosis, the combination of ibrexafungerp and isavuconazole has shown synergy by improving survival rates, reducing pulmonary tissue damage, and decreasing fungal biomarkers in persistently neutropenic rabbits ( Petraitis et al. 2020 ). A Phase 3 open-label study in adult patients with refractory or intolerant fungal infections, including invasive pulmonary aspergillosis, reported a 46.2% success rate for ibrexafungerp ( NCT03059992 ). The inclusion of patients with biofilm-associated infections in this study suggests that ibrexafungerp’s efficacy may extend to these challenging cases, supporting its potential role in clinical practice. With the rise of antifungal resistance in A. fumigatus , particularly in biofilm-related infections, novel therapeutic agents such as olorofim, opelconazole, and ibrexafungerp have emerged as crucial alternatives. Continued investigation through rigorous clinical trials remains critical to validate the efficacy and safety of these innovative antifungal agents. In this review, we have summarized the pharmacokinetics, dosing, and safety profiles of the emerging novel antifungals ( Table 1 ). While these compounds show great potential for expanding the antifungal pipeline, translating them into safe and effective clinical therapies remains a significant challenge. One of the key challenges is the main biological and biochemical pathways targeted by antifungals are conserved between humans and fungi ( Roemer and Krysan 2014 ). Thus, several agents that effectively inhibit fungal growth often display toxicity in human cells. Toxicity is further compounded by the risk of drug–drug interactions, especially in immunocompromised patients who are already on complex multi-drug regimens. Additionally, pharmacokinetic and pharmacodynamic restrictions, such as limited oral bioavailability, poor tissue penetration, or suboptimal CNS distribution, limit the translational potential of many novel antifungals in systemic or invasive fungal infections ( Felton et al. 2014 ; Ashley 2019 ; Carmo et al. 2023 ). Beyond the scientific challenges, cost and regulatory approval pose further concerns ( Agbadamashi and Price 2025 ). The development and manufacturing of novel antifungals can be prohibitive, requiring hundreds of millions of dollars and over a decade to reach approval ( Fisher et al. 2022 ; Schinas et al. 2024 ), particularly for natural products that require complex production pipelines. Collectively, these challenges highlight the importance of integrating pharmacological, clinical, and economic considerations early in antifungal drug development. To overcome these challenges, innovative approaches and trial designs are required to develop novel antifungals. To strengthen translational insight, we categorized antifungals based on which stage of the biofilm lifecycle they mainly affect such as adhesion, germination, or maturation. Agents that interfere with cell adhesion or early metabolic activity can prevent biofilm formation. Due to their fungistatic activity, azoles such as fluconazole and next-generation tetrazoles, such as oteseconazole, and VT-1598, inhibit biofilm formation at the adhesion stage of biofilm by inhibiting ergosterol synthesis, which impairs the membrane integrity and reduces surface colonization ( Tits et al. 2020 ). Once biofilms are formed, agents must penetrate or disrupt the protective ECM. Amphotericin B and echinocandins are the most effective against mature biofilms, but their efficacy is reduced ( Martinez and Casadevall 2006 ). Emerging agents such as glucan synthase inhibitors are more promising, which are active against mature fungal biofilms, and turbinmicin, which targets the vesicular transport pathway and penetrates the ECM ( Zhao et al. 2021 ; Ceballos-Garzon et al. 2025 ). Drug repurposing offers a promising strategy for discovering effective antifungal drugs, particularly against biofilm-associated infections caused by C. albicans , C. neoformans , and A. fumigatus . This approach is advantageous due to its cost-effectiveness and the availability of established toxicity parameters for most of the drugs in human use. By leveraging high-throughput screening and exploring the synergistic potential of existing drugs, researchers can expedite the discovery of antifungal treatments ( Table 2 ). Biofilm-associated C. albicans infections, often linked to catheter use, are notably resistant to conventional antifungal therapies. Repurposing existing drugs has shown promising results in overcoming these challenges. Ebselen, a synthetic organoselenium compound under investigation for hearing loss, Menire’s disease, SARS-CoV-2, and other diseases, has demonstrated significant antifungal potential. It protects against reactive oxygen species (ROS) cellular damage by acting as a glutathione peroxidase mimetic ( Zhou et al. 2017 ). In addition to its antioxidant effects, it functions as a neuroprotectant by neutralizing free radicals. Notably, ebselen effectively inhibits C. albicans biofilm growth at 8.96 μg/mL and has demonstrated similar activity against non-albicans Candida species such as the multidrug-resistant C. auris ( Wall et al. 2018 ). Cyclosporin A, an immunosuppressant that inhibits the synthesis of IL-2 ( Tapia et al. 2025 ), restores fluconazole efficacy and enhances C. albicans biofilm inhibition ( Marchetti et al. 2000 ). Similarly, pitavastatin, a cholesterol-lowering statin that inhibits HMG-CoA reductase, demonstrates synergy with fluconazole against biofilms, suggesting its antifungal properties primarily target biofilm formation ( Eldesouky et al. 2020 ). Interestingly, serotonin reuptake inhibitors, including sertraline and fluoxetine, also exhibit anti-biofilm activity both alone and in combination with fluconazole, providing another potential avenue for overcoming antifungal resistance ( Gu et al. 2016 ; Rodrigues et al. 2023 ). Collectively, these findings showcase the value of drug repurposing as a practical and cost-effective strategy to enhance therapeutic options against resilient biofilm-associated C. albicans infections. Repurposed drugs have also been tested in mouse models and demonstrated biofilm reduction against C. albicans infection. In a mouse catheter model, auranofin, a gold-containing anti-inflammatory used to treat rheumatoid arthritis, significantly reduced C. albicans biofilms at 3 and 10 μg/mL ( Felix et al. 2023 ). It also exhibits synergistic interaction against mature C. albicans biofilms, when combined with pentamidine, an anti-protozoal drug that disrupts nucleic acids and protein synthesis ( Lin et al. 2022 ). Beyond auranofin, other repurposed drugs identified through this high-throughput screening include: Avermectin, Benzbromarone, Yohimbine hydrochloride, Zotepine, and Pyrvinium pamoate, all of which demonstrated anti- C. albicans biofilm activity ( Siles et al. 2013 ). Targeting mitochondrial function, NDU1, a component of Complex I in fungal respiration, has emerged as a novel fungal target ( Sutar et al. 2022 ). Niclosamide, an FDA-approved anti-helminthic, inhibits NDU1 and disintegrates mature biofilms at 2 μg/mL ( Sutar et al. 2022 ). Its nanoparticle form penetrates ECM, disrupting hyphal structures critical to biofilm. Interestingly, niclosamide nanoparticles reduced the fungal burden of the tongue, and vagina in the mouse models of oropharyngeal candidiasis and vulvovaginal candidiasis, respectively ( Sutar et al. 2022 ). Such NDU1 inhibitors are examples of the importance of finding compounds and strategies that can attack and get around the main line of biofilm defense, its protective ECM. The management of C. neoformans biofilms remains a significant challenge due to their enhanced resistance to standard antifungal therapies. Drug repurposing has identified several non-antifungals with potent activity against C. neoformans biofilms, offering new avenues for therapeutic intervention. Among these, antituberculosis drugs, such as isoniazid and ethionamide, have shown to reduce cryptococcal biofilm metabolic activity by approximately 50%. Additionally, a synergistic effect was observed when isoniazid and ethionamide was combined with fluconazole or itraconazole, leading to a substantial reduction in metabolic activity, and greater efficacy against mature biofilms, compared to either azole alone ( Cordeiro Rde et al. 2015 ). Mebendazole, an antiparasitic, has also demonstrated potent anti-cryptococcal activity. It significantly reduces cryptococcal biofilm formation and eradicate mature biofilms at 0.0156 μM ( Joffe et al. 2017 ). Furthermore, mebendazole, when used in combination with amphotericin B, exhibited anti-cryptococcal activity at lower MIC for both compounds ( Joffe et al. 2017 ). Antipsychotic drugs have demonstrated antifungal activity against C. neoformans. Specifically, haloperidol, primarily used against schizophrenia and Tourette’s syndrome, possesses antifungal activity. In particular, the haloperidol derivative compound B10 potently reduces cryptococcal biofilm formation by ≥80% at 2 μg/mL, and interacts synergistically with fluconazole, significantly reducing the brain fungal load in mice ( Ji et al. 2020 ). These findings underscore the potential of drug repurposing as an efficient strategy to combat C. neoformans biofilms, offering a promising pathway to expand the current antifungal arsenal with reduced development time and cost. The rising prevalence of triazole-resistant A. fumigatus strains, particularly those capable of forming robust biofilms, emphasizes the critical need for alternative therapeutic strategies. Sodium new houttuyfonate (SNH), derived from houttuynin, traditionally used in Asia for its antimicrobial properties. SNH’s interferes with the A. fumigatus cell steroid synthesis pathway, ultimately inhibiting ergosterol synthesis ( Zhang et al. 2022 ). XTT reduction assays demonstrated that SNH disrupts A. fumigatus mature biofilms, reducing it by ~20% at 1× MIC and ~80% at 4× MIC. Microscopic analysis showed clear changes, including shorter fungal threads and thinner biofilms ( Zhang et al. 2022 ). Moreover, SNH-treated mice with aspergillosis showed a significant reduction in fungal burden, tissue damage, and proinflammatory cytokine levels across multiple organs. Alexidine dihydrochloride (AXD), a bis-biguanide compound, has shown anti-biofilm activity against C. albicans, C. neoformans , and A. fumigatus ( Kim et al. 2020 ). Specifically, AXD completely disrupted A. fumigatus mature fungal biofilms at 1.5 to 6 μg/mL, by inhibiting fungal filamentation and proliferation ( Kim et al. 2020 ). Additionally, miltefosine, widely used in the treatment of leishmaniasis, has displayed fungicidal activity against A. fumigatus . Its mechanism involves disruption of sphingolipid biosynthesis, and it is effective against azole-resistant clinical isolates. This is supported by transcriptomic analysis, which identified a miltefosine-sensitive transcription factor (SmiA) that regulates sphingolipid pathway genes. Moreover, sphingolipid profiling revealed reduced levels in miltefosine-treated mutants ( Dos Reis et al. 2021 ). However, studies have yet to specifically examine miltefosine’s efficacy against fungal biofilms. Finally, spiro-acridine derivatives, better known for their antile-ishmanial and antitumoral activities, have been identified as potential inhibitors of fungal chitinases. In particular, compound 5 strongly inhibits chitinases in A. fumigatus , suggesting potential future exploration as biofilm-targeted antifungal therapies ( de Oliveira Viana et al. 2023 ). Repurposed drugs offer a faster, cost-effective strategy against fungal biofilms, with potential for synergy when combined with existing antifungals and reduced host toxicity. The continued exploration of these compounds for their antifungal properties and their synergistic effects, alongside improved delivery methods show great promise for improving the management of biofilm-related infections and overcoming the growing threat of antifungal resistance. Natural products have historically been a rich source of antifungal discovery, providing unique chemical diversity and mechanism of action often absent in synthetic compounds. The search for natural products has gained momentum as resistance to existing treatments continues to rise. Recent studies have investigated a variety of substances derived from plants, microbes, and marine organisms, revealing promising antifungal activities and potential for new therapeutic strategies ( Table 3 ). Vitamins have emerged as accessible anti-biofilm agents. Vitamin D3 reduces C. albicans biofilm formation by up to 88% at 60 μg/mL, with an IC50 of 7.5 μg/mL, interfering with ribosome biogenesis, and metabolic pathways ( Kherad et al. 2023 ). It not only reduces C. neoformans biofilm metabolic activity at 0.4–0.8 mg/mL, but also prevents adhesion, limiting biofilm development ( Huang et al. 2023 ). Additionally, Vitamin C or ascorbic acid, has shown anti-biofilm effects though its mechanism differs from Vitamin D ( Chen et al. 2024 ). Essential oils exhibit strong antifungal properties against C. albicans and C. neoformans biofilms. Thymol and carvacrol, found in thyme and oregano oils, significantly reduce biofilm adherence and metabolic activity ( Kumari et al. 2017 ; Hacioglu et al. 2021 ; Shariati et al. 2022 ; Vassiliou et al. 2023 ). Moreover, combining essential oils with antifungals has shown to enhance the antifungals’ efficacy against C. albicans . For example, Eucalyptol, the active component of eucalyptus oil, acts in synergy with amphotericin B, nystatin, and itraconazole by reducing their MICs against C. albicans biofilms ( Keymaram et al. 2022 ). Other essential oils, such as coconut, tea tree, and clove oil, have demonstrated anti- C. albicans biofilm properties ( Francisconi et al. 2020 ; Dudek-Wicher et al. 2022 ; Hou and Huang 2024 ). Plant-derived compounds, such as curcumin, piperine, and ginger, demonstrate anti-biofilm activity against C. albicans biofilms ( Tsopmene et al. 2024 ). However, achieving such concentrations in vivo is unlikely due to their poor pharmacokinetic profile, with low plasma levels and a short elimination half-life in human subjects ( Zick et al. 2008 ). Other herbs and natural extracts with antifungal activity against C. albicans biofilms include garlic, cinnamon, pomegranate, and cranberries ( Shuford et al. 2005 ; Veilleux and Grenier 2019 ; Celiksoy et al. 2022 ). Chitosan, a fibrous sugar that is found in the exoskeleton of crustaceans, has garnered significant interest for its activity against fungal pathogens. Chitosan at 2.5 mg/mL significantly reduces the metabolic activity of C. neoformans biofilms by 40% ( Martinez et al. 2010 ). While effective in vitro , this concentration is relatively high and may not be therapeutically feasible for clinical use due to solubility limitations and potential toxicity. Consequently, chitosan is more likely to be useful in topical or localized applications, such as coatings for medical devices or wound dressings. Chinese medicine has also been used to treat C. albicans and C. neoformans biofilms ( Qian et al. 2022 ). For instance, artemisinin, an FDA-approved drug used as an anti-malarial drug ( Heller and Roepe 2019 ), inhibited hyphal formation by targeting biofilm-related genes of C. albicans ( Sumlu et al. 2024 ). Paeonol comes from the root bark of Paeonia suffruticosa and acts as anti-inflammatory ( Wu et al. 2021 ). Paeonol inhibits the hyphae formation of C. albicans , disrupts C. neoformans mature biofilms at 4 and 8 mg/mL, and impairs C. neoformans capsule and melanin production, suggesting multifaceted anti-virulence mechanism of action ( Qian et al. 2022 ). Antifungal peptides have emerged as effective agents to fight fungal infections, including C. neoformans . SP1 antifungal peptide, derived from Saccharomyces cerevisiae , was shown to reduce the biomass of the proliferating C. neoformans biofilm by 86.4% and its metabolic activity by 96.9% at 16 μM, while also reducing the metabolic activity of mature biofilms by 73.6% at 64 μM ( Liu et al. 2023 ). AMP-17, a peptide derived from Musca domestica , which is the most common house fly, demonstrated efficacy against C. neoformans biofilms. At 64 μg/mL, AMP-17 decreased biofilm biomass by 78% and reduced metabolic activity by 86% ( Yang et al. 2022 ). These findings highlight the potential of searching for new peptides with anti-cryptococcal biofilm activity. Targeting heat shock proteins (Hsp), particularly Hsp90, represents a novel approach to combat fungal infections ( Fu et al. 2022 ; Fang et al. 2025 ). Hsp90 functions as a virulence factor, contributing to both antifungal resistance and the pathogenicity of C. neoformans ( Fang et al. 2025 ). Specifically, it plays a role in secretion of the polysaccharide capsule which is critical for biofilm formation. Radicicol, a natural compound isolated from the Fusarium species, is a potent inhibitor of Hsp90, with MIC values of 1–2 μg/mL against planktonic C. neoformans ( Cordeiro et al. 2016 ). While radicicol alone showed no activity against fungal biofilms, it demonstrated synergistic effects when combined with fluconazole or itraconazole, inhibiting both mature biofilms and biofilm formation ( Cordeiro et al. 2016 ). This highlights the potential of Hsp90 inhibitors as adjuvants to enhance the efficacy of existing antifungal therapies against cryptococcal biofilms. Plakortide F acid (PFA), a marine-derived polyketide endoperoxide, has demonstrated strong inhibitory activity against A. fumigatus by disrupting calcium homeostasis, leading to cell death ( Xu et al. 2011 ). Additionally, PFA was found to activate the calcineurin-dependent response element, highlighting its interference with key calcium signaling pathways in yeast ( Xu et al. 2011 ). Cis-9-hexadecenal is a plant-derived substance with activity against A. fumigatus biofilms. Cis-9-hexadecenal inhibited growth at 0.078 mg/mL, and exhibited significant anti-biofilm activity at 0.156 mg/mL ( Hoda 2019 ). Scanning electron microscopy showed A. fumigatus extensive biofilm disruption, characterized by the absence of ECM and hyphal damage, outperforming amphotericin B. Cytotoxicity studies indicated low toxicity toward human lung epithelial cells (L-132), suggesting a favorable safety profile. Given its antifungal and antibiofilm properties, cis-9-hexadecenal warrants further investigation as a potential therapeutic agent against A. fumigatus biofilm-associated infections. Natural products are still a vastly unexplored resource that holds much potential in combating fungal infections. Although natural products may have low antifungal potency when used alone, they can serve as adjunct therapies to enhance the effectiveness of current antifungals, and strengthen the immune response, hence adding another protective dimension against biofilm-related infections. The treatment options against fungal biofilms are not solely limited to the use of antifungal drugs and natural products as there is extensive work being done on alternative options that showcase potential. Photodynamic therapy uses a photosensitizing agent activated by a specific wavelength of light with the goal of generating ROS, which results in microbial cell damage and death. Two notable photosensitizers include riboflavin (Vitamin B2) and hypericin, which are an antraquinone derivative found in Hypericium perforatum . Riboflavin and hypericin are activated by blue and yellow-orange light, respectively and have demonstrated significant efficacy in reducing the viability of fungal biofilm formation ( Łopaciński et al. 2024 ). Further research is required on this technique as the nature of generating ROS generates toxicity concerns. Alteration of surfaces used on medical devices is a crucial to prevent fungal colonization. For instance, silver-camphorimine treatment on stainless steel medical devices has been shown to inhibit the colonization of C. albicans biofilms by ~82%, without mammalian toxicity ( Pinheiro et al. 2021 ). Graphene coatings on titanium implants has also been shown to significantly reduce fungal adherence ( Morin et al. 2025 ). Other coatings that prevent fungal biofilm formation on medical devices include the previously mentioned chitosan on polyurethane intravenous catheters and imidazolium salt conjugated poly(L-lactide) ( Vera-Gonzalez and Shukla 2020 ). The prevention of fungal biofilms can also be achieved through surface modifications tethered with clinically used antifungals, novel molecules, and peptides ( Vera-Gonzalez and Shukla 2020 ). For example, caspofungin tethered with titanium disks has shown complete inhibition of fungal attachment, compared to titanium alone ( Kucharíková et al. 2016 ). Additionally, β-cyclodextrin has been applied with miconazole in medical devices, which resulted in ~90% reduction of C. albicans biofilm ( Nava-Ortiz et al. 2010 ). Due to high resistance profile of current antifungals, small molecules and peptides have been used to prevent fungal biofilms. Filastatin, a small molecule that inhibits C. albicans adhesion, was applied on various materials including bioactive glass (cochlea implants), dental resin (dentures), and silicone (catheters), resulting in 99%, 77% and 60% C. albicans biofilm formation reduction, respectively ( Vargas-Blanco et al. 2017 ). Vaccines are an alternative approach developed to target specific antigens involved in biofilm formation or trigger a broad immune response against the fungal cell wall. NDV-3A, a novel vaccine in phase 2 clinical trials for the treatment of fungal infections, has induced the production of anti-rAls3p-N antibodies. These antibodies interfere with C. albicans adhesion to and invasion of endothelial cells and protect against biofilm formation in a central venous catheter mouse model ( Alqarihi et al. 2019 ; Singh et al. 2019 ). Additionally, fungal cell wall carbohydrates are attractive targets for immunotherapy. Glycoconjugate vaccines and monoclonal antibodies are directed against β-glucans and mannans, with preclinical studies showing protective efficacy and identifying several antibody candidates for development ( Devi 1996 ; Torosantucci et al. 2005 ; Komarova et al. 2018 ; Del Bino and Romano 2020 ). Despite this, there are no approved vaccines available to prevent fungal infections or biofilms in clinical use. Current efforts focus on developing vaccines that can protect against fungal infections ( Del Bino and Romano 2020 ). Catheter-related bloodstream infections pose a significant healthcare challenge ( Alshahrani et al. 2023 ), with fungal infections, particularly those caused by Candida , are the third most common catheter-associated infection ( Pierce et al. 2015 ). These infections cause high mortality rate, with 25% of patients with candidemia die during hospitalization ( Center for Disease Control and Prevention 2024 ). Additionally, it is extremely difficult to eradicate these infections by the removal of the medical device due to clinical reasons. To address this, antifungal lock therapy has emerged as a potential strategy, which involves adding high concentrations of antifungals into the catheter lumen to eradicate infections ( Walraven and Lee 2013 ). While preclinical studies support the efficacy of antifungal lock therapy, there is limited comparative or clinical data to establish standardized recommendations for its use in patient management. The antifungal therapies discussed in this review represent diverse approaches, ranging from novel antifungals to combinational therapies, and nanotechnology-based strategies. Among the emerging antifungals, the most promising therapies are rezafungin, fosmanogepix, ibrexafungerp, olorofim, and turbinmicin ( Schinas et al. 2024 ). Those agents are also becoming successful as they either directly or indirectly inhibit biofilm formation or penetrate ECM, with novel mechanisms that bypass existing resistance, and strong efficacy without rapid resistance emergence. When compared, these agents display several advantages. Ibrexafungerp, olorofim, fosmanogepix, and oteseconazole offer oral bioavailability and reduced toxicity, rezafungin enables long-acting intravenous therapy, and turbinmicin introduces a novel mechanism by disrupting extracellular vesicle-mediated ECM assembly. Combinational therapy has been the first line of treatment of several diseases such as malaria, tuberculosis, and HIV ( Eastman and Fidock 2009 ; Bock and Lengauer 2012 ; Zumla et al. 2012 ; Spitzer et al. 2017 ). It has also emerged as a highly effective strategy against fungal pathogens by pairing conventional antifungals with co-drugs/novel antifungals that potentiate their activity. For example, several compounds can enhance the antifungal activity of azoles, especially fluconazole, by converting its fungistatic activity to fungicidal, often through the inhibition of efflux pumps ( Maesaki et al. 1998 ; Onyewu et al. 2003 ; Liu et al. 2022 ). Similarly, they can restore the efficacy of amphotericin B in resistant strains by restoring its activity and reduce its toxicity by lowering its dose ( Salama et al. 2024 ; Elgammal et al. 2025 ). Moreover, combination therapy can also delay the emergence of resistance by attacking multiple fungal targets, improving patient outcomes in biofilm and invasive infections. Despite these scientific advances, significant gaps persist. Some emerging therapies have limited efficacy against mature biofilms or in central nervous system infections, where pharmacokinetics and tissue penetration present major barriers. Resistance suppression is promising for agents with novel targets, but cross-resistance and durability of efficacy require further validation.