Bioprospecting of fungi for therapeutic enzymes has emerged as a promising field in biotechnology, leveraging the diverse enzymatic capabilities of fungi to address various medical challenges. Fungal enzymes have significant potential for large-scale production at low cost and with high purity because of their ability to be produced extracellularly in large amounts. In addition, the unique properties of fungal enzymes, such as their ability to undergo posttranslational modifications and break down complex substrates and their high yield, stability, robustness, and reduced immunogenicity, make them promising in the therapeutic field. Enzymatic therapy is a rapidly advancing treatment option for various diseases, with numerous therapeutic enzymes such as hydrolases (amylase, protease, lipase, cellulase, etc.), collagenase, chitinase, l-asparaginase, laccase, and uricase, which are important for pharmaceutical applications. This chapter explores the mechanism, status, challenges, and considerations of fungal enzymes used in thrombolytic, digestive aid, enzyme replacement therapy (ERT), wound care management, and anti-inflammatory, anti-cancer, and anti-bacterial activities.