We screened for bacterial phospho-N-acetylmuramyl-pentapeptide-translocase (MraY: EC 2.7.8.13) inhibitors with the aim of discovering novel antibiotics and observed inhibitory activity in the culture broth of an actinomycete, SANK 60501. The active compounds, muraminomicins A, B, C, D, E1, E2, F, G, H, and I exhibited strong inhibitory activity against MraY with IC50 values of 0.0105, 0.0068, 0.0104, 0.0099, 0.0115, 0.0109, 0.0089, 0.0134, 0.0186, and 0.0094 μg ml-1, respectively. Although muraminomicin F exhibited favorable antibacterial activity against drug-resistant Gram-positive bacteria, this activity was reduced with the addition of serum. To efficiently supply the core component for chemical modification studies, production was carried out in a controlled trial by adding myristic acid to the medium, and a purification method suitable for large-scale production was successfully developed.