There is a growing concern in using zinc oxide nanoparticles (ZnO NPs) for medical devices as alternative options in reducing hospital-acquired infections (HAIs). The commensal HAIs; Staphylococcus aureus (S.aureus) infect patients and lead to increased rates of morbidity and mortality. This study aims to investigate the antibacterial action of ZnO NPs in three different shapes; nanorod, nanoflakes and nanospheres impregnated in low-density polyethylene (LDPE) against S.aureus ATCC 25923. Methods: The antibacterial efficiency of ZnO NPs was studied through two standard test methods included were based on Clinical Laboratory Standards Institute (CLSI) guidelines MO2-A11 under light conditions of 5.70 w/m2 and American standard test method (ASTM) E-2149. Results: Preliminary screening did show a significant growth inhibition against S.aureus with ZnO NPs nanorod and nanoflakes, approximately in 7 to 8 mm zones of inhibition. Further analysis using ASTM E-2149 in dynamic conditions revealed variable activity depending on incubation treatment periods. It demonstrated the ZnO NPs in nanoflakes and nanosphere shape showed better inhibition against S.aureus with maximum reduction (100%). The FESEM results strongly suggest that the structure of ZnO nanoflakes and nanosphere played an importance role in nanomaterial-bacteria interaction which consequently cause cell membrane damage. Additionally, the irradiation under light treatment also enhance the generation of ROS and free radicals which helps the bactericidal activity against S.aureus. Conclusion: This study provides new insights for the antibacterial action of ZnO NPs/LDPE thin films in future biomedical appliances to reduce HAIs risks.