Background: The spread of pathogenic microorganisms in food and beverage and their resistance to antibiotics have raised major concerns for public health. The aim of this study was to investigate the antimicrobial activity of various metal oxide nanoparticles (NPs) including zinc oxide (ZnO), magnesium oxide (MgO), and iron oxide (Fe₂O₃) NPs against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Furthermore, the antimicrobial activity of these NPs in milk was studied along with mild heat. Methods: In this experimental study, the antibacterial activity of ZnO, MgO, and Fe₂O₃ NPs were initially evaluated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) methods. Later, the antimicrobial effect of these NPs was investigated in milk along with mild heating. To determine the morphological changes in S. aureus and E. coli, electron microscopy scanning was applied before and after the antimicrobial treatments. Results: The MBC and MIC values presented by Fe₂O₃, ZnO, and MgO NPs against pathogenic bacteria showed that MgO NPs were the most potent substances for inhibiting the growth of S. aureus and E. coli. The results also indicated that use of these NPs had synergistic effects in combination with the heating treatment. Electron microscopy scanning also revealed that treatment with MgO NPs could distort and impair the cell wall of the pathogenic bacteria, leading to the leakage of intracellular components and bacterial death. Conclusion: The results suggest that MgO, ZnO, and Fe₂O₃ NPs can be applied for industrial food processing as effective antimicrobial compounds to decrease the temperature required for pasteurizing milk