Until recently, little was known about the fungi found in shark gills and their biomedicinal potential. In this article, we described the isolation, bioactivity, diversity, and secondary metabolites of bioactive fungi from the gill of a shark ( Carcharodon carcharias ). A total of 115 isolates were obtained and grown in 12 culture media. Fifty-eight of these isolates demonstrated signifi cant activity in four antimicrobial, pesticidal, and cytotoxic bioassay models. Four randomly selected bioactive isolates inhibited human cancer cell proliferation during re-screening. These active isolates were segregated into 6 genera using the internal transcribed spacer-large subunit (ITS-LSU) rDNA-sequence BLAST comparison. Four genera, Penicillium , Aspergillus , Mucor , and Chaetomium were the dominant taxa. A phylogenic tree illustrated their intergenera and intragenera genetic diversity. HPLC-DAD-HRMS analysis and subsequent database searching revealed that nine representative strains produced diverse bioactive compound profi les. These results detail the broad range of bioactive fungi found in a shark's gills, revealing their biopharmaceutical potential. To the best of our knowledge, this is the fi rst study characterizing shark gill fungi and their bioactivity.