During the process of optimization of pargamicin A (PRG-A) production, we discovered new active components PRG-B (I, R1=R2=H, R3=Et), PRG-C (I, R1=OH, R2=H, R3=Et), and PRG-D (I, R1/R2=carbonyl, R3=Et) in the culture broth of the PRG-A-producing strain Amycolatopsis sp. ML1-hF4.The present study investigates the fermentation, isolation, structural elucidation, and antimicrobial activities of these new PRG-A analogs and discusses the structure-activity relation of PRGs.PRG-A and PRG-C exhibited potent antibacterial activity against Gram-pos. bacteria including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis/faecium; the antibacterial activity of PRG-B and PRG-D against these bacteria was weaker.PRG-C and PRG-D, which possess a polar group in the northern region of Pip, exhibited 4-8-fold weaker antibacterial activity against staphylococci than against enterococci, whereas PRG-A and PRG-B showed equal activity against staphylococci and enterococci.