A new antimycin group antibiotic, antimycin A9, was isolated from a cultured broth of Streptomyces sp. K01-0031 together with antimycins A3a, A3b, A4, and A7, and flazin methyl ester. Antimycin A9 is the first antimycin having an aromatic 8-acyl residue. It showed potent nematocidal and insecticidal activities against Caenorhabditis elegans and Artemia salina, respectively. It inhibited bovine heart NADH oxidase at nanomolar level like other known antimycins.