Pulvomycins B-D (1-3) were discovered from an estuarine Streptomyces strain along with the known pulvomycin (4). The 22-membered macrocyclic lactone structures of 1-3 were determined based on the interpretation of NMR, UV, and MS data, the modified Mosher's method, and Kishi's bidentate chiral solvent NMR spectroscopy. Genomic analysis of the bacterial strain revealed the biosynthetic gene cluster of pulvomycin and enabled us to propose the trans-acyltransferase polyketide biosynthetic pathway. Pulvomycin D displayed potent cytotoxic activity against various cancer cell lines.