Fungi form biofilms by attaching to surfaces and forming a protective matrix of extracellular polymeric substances (EPS), resulting in resilient microbial communities that can withstand environmental stresses and evade host immune system attacks (Malinovská et al, J Fungi 9:955, 2023). EPS comprises macro- and micromolecules that perform numerous functions, including adhesion, cell aggregation, protection against antimicrobial agents and host defense, provision of nutrients, stabilization of cell communities, and facilitation of genetic exchange. Different biology, environmental conditions, physical stress, and nutrient availability cause cells to secrete different components and proportions of EPS, leading to significant differences in the composition, three-dimensional structure, and functionality of the biofilm substrates formed. The protective nature of EPS makes combating biofilm-associated infections a particular challenge. In addition, EPS production enhances adhesion to the cell surface and promotes the retention of certain heavy metals, further complicating treatment strategies (Wang et al, Heliyon 10:e32766, 2024). Biofilms play a crucial role in the pathogenesis of fungal infections as they increase fungal resistance to antifungal treatments and contribute to persistent infections. Biofilms play a crucial role in various applications, including biotechnology, where they can be used for bioremediation and wastewater treatment. Understanding biofilm dynamics is crucial for developing effective antifungal therapies, as biofilm-associated infections often resist conventional treatments (Ali et al, Microorganisms 11:1934, 2023). In this chapter, basic protocols for the formation of fungal biofilms in a 12-well plate are described. The fungi are cultured in special media to promote biofilm formation. The process begins with the attachment of the fungi to the surface of the well, followed by the secretion of EPS to form the biofilm matrix. In subsequent steps, the biofilm must be maintained under favorable growth conditions, which typically includes optimizing temperature and nutrients to ensure robust biofilm development. To study the effects of antifungal drugs on biofilm formation, the biofilm is exposed to the drug at different concentrations. Treatment with the drug aims to disrupt the architecture of the biofilm and prevent further development. The disruption of the biofilm is quantified using crystal violet staining, a widely recognized method for measuring the biomass of the biofilm. Crystal violet binds to the EPS and cellular components of the biofilm, allowing visualization and quantification of biofilm formation. After staining, biofilm biomass is assessed by measuring the absorbance of the dye, providing valuable insight into the effectiveness of the antifungal treatment in disrupting biofilm formation. The data obtained can provide important information on how different drugs affect the structure of the biofilm and its resistance to antifungal treatments. These findings are crucial for a better understanding of fungal biofilms and for the development of more effective strategies to combat fungal infections caused by biofilms. This chapter, makes an important contribution to mycology and antifungal therapy by providing a detailed protocol for biofilm formation and disruption. The knowledge gained from such studies is essential to improve the treatment of fungal infections and to develop new therapeutic approaches to effectively combat biofilms.