PF-1163A, a depsipeptide antifungal compound isolated from Penicillium, blocks ergosterol synthesis with an IC50 of 12 ng/ml. This compound specifically inhibits the enzyme ERG25p, a C-4 methyl oxidase in the ergosterol biosynthetic pathway, leading to its antifungal activity. Notably, in Saccharomyces cerevisiae modified to express ERG biosynthesis genes, PF-1163A exhibits an inhibition concentration (MIC value) of 12.5 µg/ml against ERG25p, though strains overexpressing ERG25p show resistance. Additionally, PF-1163A effectively suppresses the growth of Candida albicans (MIC = 8 µg/ml), without affecting other Candida species, Aspergillus fumigatus, or HepG2 cells. Remarkably, it enhances the efficacy of fluconazole against azole-resistant C. albicans strains, showing a significant decrease in MICs (1 and 0.0078 µg/ml alone and in combination, respectively). PF-1163A distinguishes itself from PF-1163B by its more polar nature.