With the aim of developing environment-friendly agricultural products with antifungal activity againstFusarium oxysporumf. sp. lycopersici, a causative agent of Fusarium wilt, active substances fromBoesenbergia pulcherrimaroots were isolated. The hexane fraction fromB. pulcherrimaroot extract was analyzed by GC/MS. The main peaks were estimated and identified to be methyl eugenol, methyl isoeugenol, elemicin, alpha-asarone, and 1,2-dimethoxy-4-(2-methoxyethenyl)benzene based on the Wiley library and by comparing retention times and mass spectra with their corresponding standards using GC/MS. For the identification of the compound in peak D that was estimated to be 1,2,4-trimethoxy-5-vinylbenzene, for which no reference standard was available, the hexane fraction was processed by column chromatography before NMR analysis. The result confirmed the compound to be 1,2,4-trimethoxy-5-vinylbenzene. Almost all compounds showed antifungal activity againstF. oxysporumbased on bioassays, and alpha-asarone had the highest activity. Therefore,B. pulcherrimaroot extract can be a potential source of environment-friendly agricultural products with antifungal activity againstF. oxysporum.