WAP-8294A2 (lotilibcin, 1) is a potent antibiotic with superior in vivo efficacy to vancomycin against methicillin-resistant Staphylococcus aureus (MRSA). Despite the great medical importance, its molecular mode of action remains unknown. Here we report the total synthesis of complex macrocyclic peptide 1 comprised of 12 amino acids with a β-hydroxy fatty-acid chain, and its deoxy analogue 2. A full solid-phase synthesis of 1 and 2 enabled their rapid assembly and the first detailed investigation of their functions. Compounds 1 and 2 were equipotent against various strains of Gram-positive bacteria including MRSA. We present evidence that the antimicrobial activities of 1 and 2 are due to lysis of the bacterial membrane, and their membrane-disrupting effects depend on the presence of menaquinone, an essential factor for the bacterial respiratory chain. The established synthetic routes and the menaquinone-targeting mechanisms provide valuable information for designing and developing new antibiotics based on their structures.