Introduction: One of the main healthproblemsis increasing resistance of bacteria to antimicrobial agents world-wide. Nanoparticles can be useful for preventing the distribution of resistant bacteria in the hospital and community. This study was carried out to determine the antimicrobial activity ofzinc oxide (ZnO) nanoparticle against isolated Staphylococcus aureus. Method of study: This study was conducted on 268 Staphylococcus aureuswhichwas isolated from healthy carriers, patients and food materials in Golestan province, innorth -east Iran. Antimicrobial activity of ZnOnanoparticles (10-30 nmdiameters) against Staphylococcus aureus was carried out by Agar dilution method and subsequently by Microdilution method for thedetermination of its minimum inhibitory concentration (MIC). Distribution of MIC were evaluated and statistically analyzed on the basis of source, antibacterialresistance, the ability of biofilm formation and virulence factors. Results: The minimum concentration of ZnO nanoparticles that could prevent the growth of isolated Staphylococcus aureus was 625 mu g/ml. MIC50 and MIC90 were 1250 and 5000 mu g/ml, respectively. The Mean MIC ofZnO nanoparticle on S.aureuswhich wasisolated from food material (2108.6 mu/ml) was more than two other groups; patients: 1614.2 mu g/ml, and Healthy Carriers: 1503.9 mu g/ml. This difference was statistically significant (P = 0.02). There was not any statistically difference in the mean ofMIC amongS.aureus which was isolated from thepatients, but the MIC value was lower in the strains which were isolated from blood cultures. The mean MICvalueof ZnO in S. aureus resistant and sensitive to Gentamicin were 1564.25 and 2343.7, respectively (p = 0.04) and in resistant and sensitive to Erythromycin were 1530.59 and 2103.4, respectively (p = 0.06). There were not any significant differences among the strains according to antibiotic resistance profiles, the ability to produce biofilm, and ager typing groups. Conclusion: In addition to size of nanoparticles and the concentration of bacteria, we found that the efficacy of ZnO nanoparticles dependent mainly on the source of Staphylococcus aureus isolation and its resistance to GentamicinandErythromycin.