Objective(s): The objective of this study was to investigate the antibacterial activities of magnesium oxide nanoparticles (MgO NP) alone or in combination with other antimicrobials (nisin and heat) against Escherichia coli and Staphylococcus aureus in milk.Materials and Methods: First, the combined effect of nisin and MgO nanoparticles was investigated in milk. Then the combined effect of nisin, heat and MgO nanoparticles was assessed in milk. Also the scanning electron microscopy was used to characterize the morphological changes of S. aureus before and after antimicrobial treatments.Results: The results showed that MgO NP have strong bactericidal activity against the pathogens. A synergistic effect of MgO in combination with nisin and heat was observed as well. Scanning electron microscopy was revealed that MgO NP treatments in combination with nisin distort and damage the cell membrane, resulting in a leakage of intracellular contents and eventually the death of bacterial cells. Conclusion: These results suggested that MgO NP alone or in combination with nisin could potentially be used as an effective antibacterial agent to enhance food safety.