The study aimed to investigate the eco-friendly preparation of zinc oxide nanoparticles (ZnO NPs) using Origanum minutiflorum extract and the inactivation effect of the ZnO NPs to Staphylococcus aureus isolates from subclinical mastitis cowmilk. In dairy farms with capacities of 30 or more, a total of 350 cows were evaluated. In a consequence of the evaluation for subclinical mastitis, 144 (41.14%) of the dairy animals were positive. Thirty-four isolates (14.70%) were identified as Staphylococcus aureus by PCR, and 29 (20.13%) of the dairy animals with subclinical mastitis were also found to be S. aureus positive. The isolates were found to be resistant to various antibiotics, including ampicillin (85.29%), penicillin (82.35%), oxacillin (61.76%), cefoxitin (58.82%), erythromycin (44.11%), vancomycin (35.29%), rifampin (38.23%), tetracycline (26.47%), ciprofloxacin (17.64%), and gentamicin (17.64%). Origanum minutiflorum -mediated ZnO NPs have been found to have antibacterial activity against all isolates, with minimum inhibitory concentration (MIC) values ranging from 2.5 to 2000 mu g/mL. The ZnO NPs were found to peak at around 350 nm in the UV -Visible analysis. Scanning electron microscopy (SEM) images confirmed the nanoparticle (NP) size and distribution of ZnO. The typical crystallite measured at 15.86 nm. Green produced ZnO NPs exhibited antioxidant activity by scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. In conclusion, an alternative antibacterial product synthesis has been provided for antibiotic substances. Because of the rising incidence of antibiotic resistance in isolates, more research on antibiotic alternatives needs to be considered.