Amentoflavone is a plant biflavonoid that was isolated from an ethyl acetate extract of the whole plant of Selaginella tamariscina (Beauv.) spring. 1D and 2D NMR spectroscopy including DEPT, HMQC, and HMBC were used to determine its structure. Amentoflavone exhibited potent antifungal activity against several pathogenic fungal strains but had a very low hemolytic effect on human erythrocytes. In particular, amentoflavone induced the accumulation of intracellular trehalose on C. albicans as a stress response to the drug, and disrupted the dimorphic transition that forms pseudo-hyphae during pathogenesis. In conclusion, amentoflavone has great potential to be a lead compound for the development of antifungal agents.