The antifungal activities and chemical composition of neem seed extract (Azadirachta indica) were investigated. An agar diffusion assay showed that neem seed extract (optimum concentration: 10%) inhibited the growth of Trametes versicolor and Gloeophyllum trabeum. The minimum inhibitory concentration and minimum fungicidal concentration were 2% and 4%, respectively, against the two fungal species. Scanning electron microscopy revealed severe morphological damage to fungal hyphae, including reduction of spores and hyphal shriveling. Fourier transform infrared spectroscopy and gas chromatography-mass spectrometry analyses identified 17 compounds that represented 80.3% of the total extract, including astaxanthin (34.3%), cinobufagin (16.9%), anodendro-side A (6.65%), 16-acetoxy-4,8,14-trimethyl-3,11-dioxo-, methyl ester (5.09%). Furthermore, T. versicolor-infected wood and G. trabeum-infected wood treated with 10% neem seed extract displayed mass decreases of 11.4 ± 5.8% and 25.8 ± 7.5%, respectively, compared with distilled water treatment (21.6 ± 4.2% and 46.9 ± 4.3%, respectively). These findings suggest the potential use of neem seed extract for antifungal wood protection.