Background: Zinc oxide nanoparticles (ZnO NPs) are known as one of the important inorganic materials used in research and health-related applications with effective antibacterial activities. Although the toxic effects of ZnO NPs have already been evaluated, more information is required to understand the possible mechanisms. Objectives: The aim of the present study was to determine the influences of pH and temperature on antibacterial activity of ZnO NPs against some strains of pathogenic bacteria. Identifying the interrelationship between toxicity and cultural conditions helps us to have a better understanding of the optimum reaction conditions for maximum antimicrobial activity. Materials and Methods: ZnO NPs were prepared and characterized and then dispersed in glycerol with the help of ammonium citrate as the dispersant. The antibacterial tests were performed by measuring the growth of Escherichia coli O157:H7 and Staphylococcus aureus with different concentrations of ZnO NPs in glycerol. All the experiments were conducted at different incubation temperatures (25-42°C) and pH levels (4-10 for E. coli O157:H7 and 5-10 for S. aureus). Results: The results showed that ZnO nanofluid have antibacterial activity against E. coli O157:H7 and S. aureus and the inhibitory effect increases with increasing the nanofluid concentration. The experiments showed that the antibacterial activity of ZnO NPs was influenced by temperature and pH. Higher antibacterial activity was observed at acidic pH levels with the maximum toxicity at pH = 4 and pH = 5 for E. coli O157: H7 and S. aureus, respectively. By raising the temperature, the toxicity of ZnO nanofluid increased, with the highest antibacterial activity at 42°C for both bacterial types in comparison with positive controls under the same conditions. Conclusions: Analysis of the results demonstrated that exposure media of ZnO NPs and cultural factors play a role in their cytotoxic effects. It could be attributed to the principal mechanism at different reaction conditions.

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