Antimicrobial peptides constitute one of the main classes of molecular weapons deployed by the innate immune system of all multicellular organisms to resist microbial invasion. A good proportion of all antimicrobial peptides currently known, numbering hundreds of molecules, have been isolated from frog skin. Nevertheless, very little is known about the effect(s) and the mode(s) of action of amphibian antimicrobial peptides on intact bacteria, especially when they are used at subinhibitory concentrations and under conditions closer to those encountered in vivo. Here we show that esculentin-1b(1–18) [Esc(1–18)] (GIFSKLAGKKLKNLLISG-NH2), a linear peptide encompassing the first 18 residues of the full-length esculentin-1b, rapidly kills Escherichia coli at the minimal inhibitory concentration. The lethal event is concomitant with the permeation of the outer and inner bacterial membranes. This is in contrast to what is found for many host defense peptides, which do not destabilize membranes at their minimal inhibitory concentrations. Importantly, proteomic analysis revealed that Esc(1–18) has a limited ability to modify the bacterium’s protein expression profile, at either bactericidal or sublethal concentrations. To the best of our knowledge, this is the first report on the effects of an antimicrobial peptide from frog skin on the proteome of its bacterial target, and underscores the fact that the bacterial membrane is the major target for the killing mechanism of Esc(1–18), rather than intracellular processes.