There is a constant need for new therapeutic compounds. Fungi have proven to be an excellent, but underexplored source for biologically active compounds with therapeutic potential. Here, we combine mycology, embryology and chemistry by testing secondary metabolites from more than 10,000 species of fungi for biological activity using developing zebrafish (Danio rerio) embryos. Zebrafish development is an excellent model for high-throughput screening. Development is rapid, multiple cell types are assessed simultaneously and embryos are available in high numbers. We found that 1,526 fungal strains produced secondary metabolites with biological activity in the zebrafish bioassay. The active compounds from 39 selected fungi were purified by liquid-liquid extraction and preparative HPLC. 34 compounds were identified by a combination of chemical analyses, including LCMS, UV-Vis spectroscopy/ spectrophotometry, high resolution mass spectrometry and NMR. Our results demonstrate that fungi express a wide variety of biologically active compounds, consisting of both known therapeutic compounds as well as relatively unexplored compounds. Understanding their biological activity in zebrafish may provide insight into underlying biological processes as well as mode of action. Together, this information may provide the first step towards lead compound development for therapeutic drug development.