This study evaluated the effect of EDTA and lysozyme on the antibacterial activities of activated ovotransferrin against 5 strains of Listeria monocytogenes. First, a disc test was performed to screen the concentrations of EDTA or lysozyme that showed antibacterial activities in ovotransferrin (O) or ovotransferrin in 100 mM NaHCO3 (OS) solution. Turbidity and viability tests were conducted using O or OS solution combined with either lysozyme (OL and OSL) or EDTA (OE and OSE). Also, OS combined with 2 mg/mL of lysozyme (OSL) or 1 mg/mL of EDTA (OSLE), or both, was applied on commercial hams to determine if the solutions show antibacterial activities on meat products. The effect of initial cell population on the antibacterial activities of ovotransferrin combined with either EDTA or lysozyme was also determined. The L. monocytogenes started to grow after 1 d of incubation in the presence of >2.0 mg/mL of lysozyme. The OL groups showed weak antibacterial activities against L. monocytogenes in brain heart infusion broth culture, and their activities were bacteriostatic. The OSL groups were bactericidal against L. monocytogenes, resulting in 1 log reduction from initial cell population. Even though OSL showed stronger antibacterial activity than OS, lysozyme had no significant effect on antibacterial activity of OS against L. monocytogenes. Also, EDTA itself at 1.0 and 2.0 mg/mL was bacteriostatic against 5 strains of L. monocytogenes. They were more susceptible to EDTA than lysozyme, and OSE1 and OSE2 had bactericidal activity against L. monocytogenes. There was a significant difference in the survivor cell populations between OS and OSE groups (P < 0.05). Therefore, EDTA enhanced the antibacterial activity of OS against L. monocytogenes. However, ovotransferrin plus either lysozyme or EDTA, or both, did not show any antibacterial effect in commercial hams during storage at 10°C. In addition, the initial population of L. monocytogenes cells influenced the antibacterial activity of OSL or OSE.