An antifungal metabolite, designated amphiol (<b>1</b>), was isolated from the culture broth of the fungus <i>Pseudogymnoascus</i> sp. PF1464. It exists as a mixture of inseparable tautomers, an acetal form and a keto form. The chemical structure was determined by spectroscopic analyses and chemical derivatization. Amphiol (<b>1</b>) showed antifungal but not antibacterial activities, while yeast mutant cells lacking ergosterol biosynthetic genes were less sensitive, implying a fungal specific, membrane-related mechanism of action.