The gastric pathogen Helicobacter pylori has developed resistance to virtually all current antibiotics; thus, there is a pressing need to develop new anti-H. pylori therapies. The goal of this work was to evaluate the antibacterial effect of oligo-acyl-lysyl (OAK) antimicrobial peptidomimetics to determine if they might represent alternatives to conventional antibiotic treatment of H. pylori infection. A total of five OAK sequences were screened for growth-inhibitory and/or bactericidal effects against H. pylori strain G27; four of these sequences had growth-inhibitory and bactericidal effects. The peptide with the highest efficacy against strain G27, C12K-2beta12, was selected for further characterization against five additional H. pylori strains (26695, J99, 7.13, SS1, and HPAG1). C12K-2beta12 displayed MIC and minimum bactericidal concentration (MBC) ranges of 6.5 to 26 microM and 14.5 to 90 microM, respectively, across the six strains after 24 h of exposure. G27 was the most sensitive H. pylori strain (MIC = 6.5 to 7 microM; MBC = 15 to 20 microM), whereas 26695 was the least susceptible strain (MIC = 25 to 26 microM; MBC = 70 to 90 microM). H. pylori was completely killed after 6 to 8 h of incubation in liquid cultures containing two times the MBC of C12K-2beta12. The OAK demonstrated strong in vitro stability, since efficacy was maintained after incubation at extreme temperatures (4 degrees C, 37 degrees C, 42 degrees C, 50 degrees C, 55 degrees C, 60 degrees C, and 95 degrees C) and at low pH, although reduced killing kinetics were observed at pH 4.5. Additionally, upon transient exposure to the bacteria, C12K-2beta12 showed irreversible and significant antibacterial effects and was also nonhemolytic. Our results show a significant in vitro effect of C12K-2beta12 against H. pylori and suggest that OAKs may be a valuable resource for the treatment of H. pylori infection.