The lipid II-binding N-terminus of nisin, comprising the so-called A/B ring system, was synthetically modified to provide antibacterially active and proteolytically stable derivatives. A variety of lipids were coupled to the C-terminus of the nisin A/B ring system to generate semisynthetic constructs that display potent inhibition of bacterial growth, with activities approaching that of nisin itself. Most notable was the activity observed against clinically relevant bacterial strains including MRSA and VRE. Experiments with membrane models indicate that these constructs operate via a lipid II-mediated mode of action without causing pore formation. A lipid II-dependent mechanism of action is further supported by antagonization assays wherein the addition of lipid II was found to effectively block the antibacterial activity of the nisin-derived lipopeptides.