ABSTRACT: ε–polylysine is a homopolymer of L-lysine, an essential amino acid, with a reportedly wide antimicrobial spectrum. This study evaluated the antimicrobial activity of ε–polylysine, as compared with known preservatives and organic acids, against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes, in culture broth. The compounds tested included ε–polylysine (0.0025% to 0.05%), sodium diacetate (0.25%), sodium lactate (3.0%), lactic acid (0.1%), and acetic acid (0.1%), alone, as well as in combination with ε– polylysine (0.0025% to 0.03%); all treatments were evaluated in tryptic soy broth supplemented with 0.6% yeast extract. Treatments were inoculated (approximately 2 log colony-forming units [CFU]/mL) with 5-strain (E. coli O157:H7, S. Typhimurium) or 10-strain (L. monocytogenes) mixtures of the pathogens. Survival/growth of the inoculated bacteria was periodically monitored during incubation at 4 °C (30 d) and 24 °C (48 h). Bactericidal effects of ε–polylysine were obtained against E. coli O157:H7 and S. Typhimurium at 4 °C. At the same temperature (4 °C), ε–polylysine alone or in combination with other compounds tested inhibited growth or was bactericidal against L. monocytogenes. All 3 pathogens were inhibited by ε–polylysine at 24 °C; however, L. monocytogenes was the most sensitive and S. Typhimurium the most resistant. The antimicrobial activity of ε–polylysine against E. coli O157:H7 and S. Typhimurium was enhanced (P < 0.05) when tested in combination with sodium diacetate or acetic acid. Combination treatments with sodium lactate resulted in loss of ε–polylysine activity by the end of the incubation period. Overall, under the conditions of this study, ε–polylysine exhibited antimicrobial effects against the 3 pathogens tested.