The rapid rise of multi-resistant bacteria poses a significant threat to our health system. The development of drugs against novel targets is urgently needed to replenish the clinical arsenal with effective antibiotics. The naturally occurring peptide thanatin kills Gram-negative bacteria by targeting the periplasmic protein bridge, in particular the lipopolysaccharide transport protein A (LptA). Using the thanatin scaffold together with phenotypic medicinal chemistry and a target-focused approach, we developed potent antimicrobial macrocyclic peptides with drug-like properties that exhibited low frequencies for development of resistance both in vitro and in vivo. Binding affinities to Escherichia coli LptA of the thanatin analogs correlated well with minimal inhibitory concentrations for both parent and thanatin-resistant E. coli strains. Mode of action studies revealed that the antimicrobial activity of the antibiotics involves the specific disruption of the periplasmic Lpt protein bridge. Our studies validate the Lpt protein bridge as a novel target for macrocyclic peptide antibiotics that can avoid current mechanisms of drug resistance. The peptides warrant further preclinical and clinical studies addressing WHO priority 1 carbapenem-resistant Enterobacteriaceae, in particular for Klebsiella pneumoniae lung infections.