A novel natural niphimycin analog, N′-methylniphimycin was isolated from the culture broth of the Streptomyces spec. 57-13. The chemical constitution was elucidated from the physico-chemical properties, NMR techniques and mass spectrometry to be a 36-membered macrolide related to azalomycin F5a, shurimycin B and RS-22C. N′-methylniphimycin displayed moderate activity against some yeasts and filamentous fungi.