Bacterial phospho-N-acetylmuramyl-pentapeptide translocase (translocase I: EC 2.7.8.13) is a key enzyme in peptidoglycan biosynthesis, and a known target of antibiotics. Here we report a novel nucleoside inhibitor against translocase I, A-94964, isolated from the culture broth of the strain Streptomyces sp. SANK 60404. A-94964 inhibited bacterial translocase I with IC50 value of 1.1 microg/ml, and showed antimicrobial activities against Staphylococcus aureus and Enterococcus faecalis with MIC of 100 and 50 microg/ml, respectively. A-94964 did not show cytotoxicity against mammalian cell lines.