Previous studies have established the potential of the oligo-acyl-lysyl (OAK) concept in generating simple chemical mimics of host defense peptides (HDPs) with improved antimicrobial properties. We investigated the antibacterial properties of such an OAK, C 16(ω7)-KK-C 12-K amide, to obtain a better understanding of the complex mode(s) of action of cationic antibacterial peptides. The average MIC, determined against a multispecies panel of 50 strains, was 6 ± 5 μg/ml. However, although the OAK exerted an essentially dose-dependent bactericidal effect (time-kill curves typically exhibited 99% death within 2 h), marked differences in the killing rates occurred among inter- and intraspecies strains. Mechanistic comparison between equally sensitive and related strains revealed death of one strain to stem from the OAK's capacity to breach the cell membrane permeability barrier, whereas the death of the related strain resulted from the OAK's direct interference with DNA functions in vivo, without detectable membrane damage. These findings therefore support the notion that the antibacterial mechanism of action of a single HDP can vary among inter- and intraspecies strains. In addition, we present data illustrating the differential effects of environmental conditions (pH, ionic strength and temperature), on the OAK's antibacterial properties, and ultimately demonstrate potency enhancement (by orders of magnitude) through selection of optimal incubation conditions. Such attributes might be useful in a variety of antibacterial applications.