The influence of NaHCO3 and citric acid on the antibacterial activity of apo-ovotransferrin in model systems and ham was investigated. The antibacterial activity of 20 mg/mL of ovotransferrin solution with added NaHCO3 (0, 25, 50, or 100 mM) or citric acid (0.25 or 0.5%) was evaluated against Escherichia coli O157:H7 and Listeria monocytogenes in brain heart infusion broth. The antimicrobial activity of ovotransferrin saturated with Fe2+ or Zn2+ against both pathogens was also measured. In addition, ovotransferrin solutions containing either 100 mM NaHCO3 or 0.5% citric acid were applied to commercial hams inoculated with E. coli O157:H7 or L. monocytogenes and stored at 4°C for 4 wk. The antimicrobial activity of ovotransferrin increased as the concentration of added NaHCO3 increased. Sodium bicarbonate (100 mM) significantly improved the antibacterial activity of ovotransferrin against E. coli O157:H7 and L. monocytogenes. Citric acid (0.5%) combined with ovotransferrin resulted in a synergistic antibacterial effect against E. coli O157:H7, and L. monocytogenes was susceptible to 0.5% citric acid alone. Sodium bicarbonate diminished the strong antibacterial activity of ovotransferrin + citric acid against E. coli O157:H7, and use of sodium citrate instead of citric acid did not produce any antibacterial activity against the pathogens. The antimicrobial activity of ovotransferrin increased significantly under acidic conditions. The Zn-bound ovotransferrin prevented the growth of L. monocytogenes as detected in the apo-ovotransferrin combined with 100 mM NaHCO3 treatment, but Fe-bound ovotransferrin had little or no inhibitory activity against E. coli O157:H7 and L. monocytogenes. Ovotransferrin + 100 mM NaHCO3 did not exhibit any antibacterial activity against the 2 pathogens in commercial hams, whereas ovotransferrin + 0.5% citric acid suppressed the growth of L. monocytogenes in irradiated hams. In conclusion, combinations of ovotransferrin with NaHCO3, citric acid, or Zn2+ enhanced the antibacterial activity of ovotransferrin against E. coli and L. monocytogenes, but there are some limitations as discussed for applying ovotransferrin to meat or meat products.