Nature-derived substances have significantly contributed to fighting against cancer for the past fifty years. Among these, the use of medicinal plants and microorganisms stands out as significant examples. Products derived from fungi constitute more than half of all clinical medications used worldwide. Over 60% of clinically approved cancer therapies originated from natural sources. Fungi represent the largest group within the eukaryotic kingdom that are capable of synthesizing numerous secondary metabolites. In recent years, numerous studies have been conducted by researchers to investigate the anti-tumor properties of secondary metabolites derived from fungus. These compounds include terpenoids, alkaloids, phenyl quinines, ligase, isocoumarin, steroids, polysaccharides, and propanoids. Notably, fungal polysaccharides have gained more attention due to their distinctive biological activities. The enzyme asparaginase extracted from strain such as Aspergillus terreus, A. niger, A. tubingensis, Trichoderma viride, and Tamaromyces pinophilis showed highest anticancer efficacy. Various genera of Penicillium also possess the significant quantity of secondary metabolites exhibiting anticancerous properties. This chapter explores the screening of various fungal secondary metabolites exhibiting cytotoxic properties, elucidating the mechanism of action in cancer therapy, and discussing the current status of fungal metabolites in clinical studies.