Fungal infections became a major medical concern as advances in medicine increased the immunocompromised population. Current antifungal therapeutics are insufficient due to their adverse side effects, limited variety, and the emergence of resistant organisms. The goal of my thesis work was to identify novel antifungal agents, which are needed to meet the increasing demand. I sought to identify compounds that target group III hybrid histidine kinases (HHK). Group III HHKs play a crucial role in stress response and virulence in pathogens across the fungal kingdom but are not found in mammals, which makes the group III HHKs an attractive drug target. I formatted a group III HHK-expressing Saccharomyces cerevisiae strain into a high-throughput bioassay and used this reporter strain to screen natural product extract and small molecule libraries. I identified several promising antifungal compounds, but my thesis focuses on the characterization of two small molecule and four natural product antifungals. All of the molecules had robust activity against numerous fungal pathogens, including drug-resistant patient isolates. They also had potent activity against Candida albicans biofilm, a drug-resistant source of bloodstream infections. Surprisingly, none of these promising drug leads directly targeted group Ill HHKs. The small molecules exerted activity by inducing oxidative and heavy metal stress, while the natural products appeared to act by depleting intracellular potassium. My thesis outlines how the screening strategy and reporter strain can be refined to streamline the identification of group Ill HHK targeting compounds.