Global health is under extreme threat due to fungal pathogens. Fungal infections lead to almost 4 million deaths per year, and the already limited arsenal of antifungals is being debilitated due to the rise in antimicrobial resistance. There is an urgent need to discover new antifungals and to explore novel environmental sources of antifungal compounds. The goal of this project was to characterize the novel antifungal activity of a component produced by the environmental bacterium Bacillus amyloliquefaciens and analyze its effectiveness in combating some of the world’s most dangerous multidrug-resistant fungal pathogens. I characterized the component’s bioactivity through evaluating its impact on fungal growth with and without current clinical antifungals at varying concentrations, assessing effects on fungal filamentation, and investigating its cytotoxicity against mammalian cells. Additionally, I performed genetic studies with heterozygous fungal strains to predict the targeted molecular function of the component. I discovered that this component, particularly in combination with the commonly used antifungal fluconazole, is extremely promising in combating infectious pathogens and shows comparatively low levels of cytotoxicity in human liver cells at effective concentrations. I identified fungal genes that are not found in humans for which reduced copy number confers hypersensitivity to the component, suggesting a mechanism of antifungal activity. This project provides a successful analysis of the novel antifungal activity of an environmental microbe-derived component with promising therapeutic potential.