Invasive fungal infections have escalated from a rare curiosity to a major cause of human mortality around the globe. This is in part due to a scarcity in the number of antifungal drugs available to combat mycotic disease, making the discovery of novel bioactive compounds and determining their mode of action of utmost importance. The development and application of chemical genomic assays using the model yeast Saccharomyces cerevisiae has provided powerful methods to identify the mechanism of action of diverse molecules in a living cell. Furthermore, complementary assays are continually being developed in fungal pathogens, most notably Candida albicans and Cryptococcus neoformans, to elucidate compound mechanism of action directly in the pathogen of interest. Collectively, the suite of chemical genetic assays that have been developed in multiple fungal species enables the identification of candidate drug target genes, as well as genes involved in buffering drug target pathways, and genes involved in general cellular responses to small molecules. In this review, we examine current yeast chemical genomic assays and highlight how such resources provide powerful tools that can be utilized to bolster the antifungal pipeline.