Abstract Background: Bioflm-related infections are difcult to be treated because of higher resistance to antimicrobial agents. Current study aims to characterize the infuence of zinc oxide nanoparticles (ZnO-NPs) on both S. aureus sus- ceptibility to antibiotics and pathogenesis. Methods: The infuence of ZnO-NPs on bioflm formation by S. aureus was characterized by the crystal violet and tube assay. The synergistic efect of ZnO-NPs in combination with antibiotics on S. aureus was characterized using the checkerboard method. The efect of ZnO-NPs on S. aureus cell surface hydrophobicity and blood hemolysis was inves- tigated. RT-qPCR was used to investigate the efect of ZnO-NPs on the expression of bioflm related genes (icaA, icaR and sarA), katA and sigB. The impact of ZnO-NPs on S. aureus pathogenesis was evaluated using mice infection model. Results: ZnO-NPs exhibited a good antibioflm activity against S. aureus. The fndings indicate a synergistic antibi- oflm efect of combination between ZnO-NPs and tested antibiotics. ZnO-NPs were capable of decreasing S. aureus cell surface hydrophobicity which could account for observed decrease in bacterial bioflm forming capacity. Moreo- ver, ZnO-NPs-treated bacteria exhibited a signifcant decrease in blood hemolysis relative to control untreated S. aureus. The expression of bioflm related genes was signifcantly repressed in ZnO-NPs treated bacteria as compared to untreated cells. Finally, the efect of ZnO-NPs on S. aureus pathogenesis was investigated using mice infection model where ZnO-NPs accelerated healing of wounds in mice as compared to control untreated mice. Conclusions: Present data support the efciency of ZnO-NPs as antibioflm agent in treatment of S. aureus infections. This study recommends the incorporation of ZnO-NPs as adjuvant with other antibiotics targeting S. aureus based on the promising fndings obtained herein in order to control infection with this pathogen. Keywords: Staphylococcus aureus, Zinc oxide nanoparticles (ZnO-NPs), Antibioflm, Pathogenesis