Fungal infections are very important to the medical community and are a cause of morbidity and mortality, particularly among the immunocompromised. Candida glabrata and Cryptococcus neoformans are two prominent fungal pathogens, causing candidiasis and cryptococcosis, respectively. Although there are now substantial genetic and genomic resources for the study of these organisms, creating and maintaining multiple knockouts in a single organism remains problematic. CRISPR-Cas9 is a novel system for genomic editing and has been adapted to many eukaryotic organisms, recently including the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Originally discovered as part of the bacterial immune system, DNA segments from invading viruses or plasmids are incorporated into the CRISPR locus. These are transcribed into RNA and direct CRISPR associated (Cas) endonuclease to complementary sequences in the invader’s genome, where Cas introduces a double stranded break. This system has been adapted to create gene disruptions or DNA insertions, as subsequent repair must alter the target site to prevent further Cas cleavage. Guide RNA (gRNA), a ~20 nt sequence specific to the gene of interest, and cloned bacterial Cas protein are introduced into prokaryotic or eukaryotic cells where they allow editing of the target gene. CRISPR-Cas9 has not yet been applied to fungal pathogens, and the aims of this study are to introduce it into the fungus Candida glabrata and Cryptococcus neoformans. Bioinformatic analysis has identified numerous gRNA target sites in multiple genes of interest across both organisms, including genes with easily scored phenotypes where we can optimize the system. Successful application of the CRISPR-Cas9 will enable highly targeted editing unhindered by marker availability in these medically important organisms.