Fungal infection is an increasingly serious clinical problem, especially in immunocompromised patients. Fungi, such as Candida albicans, Aspergillus, and Cryptococcus, can form biofilm, which enhance their drug resistance and immune evasion ability, thus become major obstacles to the treatment of infection. Biofilm formation is not limited to human body, it can also form on the surface of medical devices, leading to chronic and recurrent infection. This article elaborates the biofilm formation mechanisms and hazards caused by different fungi such as Candida albicans and Aspergillus, emphasizes immune suppression, chronic diseases, and medical devices as high-risk factors for biofilm formation. Fungi can form biofilm through multiple stages such as adhesion, proliferation, extracellular matrix (ECM) construction, and cell dispersion, and enhance drug resistance based on physical barriers and gene regulation. Although existing antifungal agents are effective in infection caused by planktonic fungi, their treatment efficacy on fungal biofilm is limited. Therefore, the article explores new treatment strategies, including non-pharmacological therapies such as photodynamic therapy and electric field therapy, as well as targeted gene editing and the application of new biomaterials. It is necessary to strengthen the combination of basic research and clinical applications, develop efficient and low toxicity treatment scheme to improve the successful treatment rate for patients.