An antifungal substance was isolated from the extract of Acorus gramineus using various chromatographic procedures. The antibiotic was identified as beta-asarone, cis-2,4,5-trimethoxy-1-propenylbenzene, on the basis of the high-resolution EI-mass, NMR, and UV spectral data. Beta-asarone completely inhibited mycelial growth of some plant pathogenic fungi, Cladosporium cucumerinum,Colletotrichum orbiculare, Magnaporthe grisea, and Pythium ultimum, in a range of 0.5-30 microg/mL. The growth of Bacillus subtilis, Erwinia carotovora subsp. carotovora, Ralstonia solanacearum, and Xanthomonas campestris pv. vesicatoria was slightly suppressed by beta-asarone. As the concentration of beta-asarone increased, M. grisea infection was drastically inhibited on rice leaves. Treatment with 500 microg/mL of beta-asarone also greatly suppressed lesion formation of Co. orbiculare on cucumber leaves. This is the first study to demonstrate in vitro and in vivo antifungal activity of beta-asarone against plant fungal pathogens M. grisea and C. orbiculare.