Bioassay-directed chromatographic fractionation of a methylene chloride extract of Ailanthus altisshima indicated the presence of 20(S), 24(R), epoxy-25-hydroxydammarane-3-one (compound 1, ocotillone) which was isolated from this plant, for the first time. Antimicrobial activity of compound 1 was measured by inhibition of bacterial and fungal cells growth and by a hemolytic assay with human erythrocytes, respectively. The results revealed that compound 1 had potent antibacterial activity against Cram-negative bacteria, P. aeruginosa and S. typhimurium, that were without hemolytic activity, whereas it had weak antimicrobial activity against Gram-positive bacteria and fungi. These results demonstrated that the compound 1 has more antibacterial activity against 6ram-negative bacteria, which have no hemolytic activity, than Gram-positive bacteria and fungi. This is the first report on the biological activities of the compound 1.