The current spreading of nanomaterial applications supports the search for further possible functions of theses diminutive particles. The antibacterial potentiality of zinc oxide (ZnO) nanoparticles (NPs) against S. aureus causing mastitis was evaluated using qualitative and quantitative assays. These tests were performed in nutrient broth and nutrient agar following standard methods. In addition, the effect of different concentrations of ZnO nanoparticles on the growth of S. aureus was measured.MIC was determined using six different concentrations of ZnO nanoparticles including 16, 8, 4, 2, 1 and 0.5 mg/ml. The MIC value S. aureus was 0.5 mg/ml. The results showed that ZnO nanoparticles have antibacterial inhibition zone of 29 mm at the concentration of 10 mg/ml against S. aureus., and the antibacterial activity of ZnO nanoparticles increased with increasing powder concentration in vitro