Water buffalo (Bubalus bubalis) are a primary source of milk in Pakistan, where bovine mastitis is a significant health issue among cattle, leading to substantial economic losses. Staphylococcus aureus is a predominant pathogen associated with mastitis; however, a detailed molecular characterization of the strains in the country remains limited. We previously characterized mastitis strains from the Hazara division of Khyber Pakhtunkhwa, Pakistan. In this study, we investigated mastitis cases in the Peshawar division, including samples from both animals and human farm workers for comparison. Higher rates of mastitis (67.27% of animals) and sub-clinical mastitis (91.03% of positive animals) were identified in Peshawar than for those (34.55% and 75.31%, respectively) previously observed in Hazara. Methicillin-susceptible S. aureus (MSSA) belonging to clonal complex 9 (ST2454) were predominant. Methicillin-resistant S. aureus (MRSA) belonging to ST22 and ST8 were also detected in the Nowshera district. While no S. aureus colonization was observed among animal handlers, evidence of hand contamination suggests a potential route for pathogen spread. Low levels of antibiotic resistance were noted amongst isolates, but higher rates were seen in MRSA. This study presents only the second comprehensive molecular investigation of S. aureus isolated from buffalo mastitis in Pakistan and indicates a concerning rise in mastitis within the province.