The antifungal activities of the crude ethanolic extract of Anacardium othonianum (Anacardiaceae) leaves (EE) and the n-hexane (HF), EtOAc (EF), n-BuOH (BF) and hydromethanolic (HMF) fractions were assayed against Candida albicans (ATCC 64548) and Trichophyton rubrum (Tr1). Additionally, the cytotoxicities were also evaluated against normal human lung fibroblasts (GM07492A). The EE yielded minimum inhibitory concentration (MIC) values of 78.12 and 312.50 µg/mL for C. albicans and T. rubrum, respectively, and no cytotoxicity was observed. The EF and BF fractions exhibited enhanced antifungal activities when compared with the MIC values obtained for the EE fraction, and no cytotoxicity was observed for either fraction. Thus, the EF fraction, which displayed the higher antifungal activity, was purified, leading to the isolation of the following compounds: amentoflavone (1), gallic acid (2), protocatechuic acid (3), and ethyl 3,4,5-trimethoxybenzoate (4). HPLC analysis confirmed the presence of compounds 1-4 and 1-3 in the EF and BF fractions, respectively, in different proportions. The results suggest that the antifungal activities of the EE, EF and BF fractions may be attributed mainly to the actions of 1, 2 and 3.