The current study intended to illustrate the influence of ZnO nanoparticles (antibiofilm agent) on the susceptibility of S. aureus to antibiotics. Furthermore, the impact of ZnO nanoparticles to S. aureus pathogenesis was also studied using murine infection model. Treatment of S. aureus biofilm related infections is highly difficult due to the higher bacterial resistance to usually used antimicrobial agents. Consequently, the findings of this study could expand our familiarity with S. aureus and so help us to control infections caused by this opportunistic microorganism. S. aureus isolated from different sources was tested for ability for biofilm formation and impact of ZnO nanoparticle to prevent biofilm formation was characterized. The results clearly showed that treating of S. aureus with ZnO nanoparticles significantly inhibit staphylococcal biofilm formation and potentiate action of commonly used antibiotics. Moreover, treating S. aureus with ZnO nanoparticles reduced expression of biofilm related genes and oxidative stress response gene. This finding shed light on the role of ZnO nanoparticles in abolishing/lowering S. aureus pathogenesis in host.