AbstractStaphylococcus aureus is a common mastitis pathogen in dairy cows, and methicillin-resistant S. aureus (MRSA) has been found in dairy farms all over the world. The study carried out on bovines from three governorates in Egypt, with the goal of determining the prevalence of MRSA in positive milk samples of subclinical mastitis, performing an antibiotic susceptibility test against MRSA isolates and determining the risk factors associated with MRSA. A total of 350 quarter milk samples (n = 200 mixed breed cow; n = 150 water buffalo) were collected and examined for subclinical mastitis using the California mastitis test (CMT) before being exposed to standard microbiological procedures for S. aureus isolation. The disc diffusion method was used to phenotypically analyse the positive S. aureus isolates for MRSA, which was verified by a PCR assay targeting the mecA gene. According to the findings of the study, 41.4% (145/350) milk samples were positive based on CMT, while 35.7% (125/350) of positive samples identified as MRSA based on PCR assay. However, the obtained results revealed non-significant disparity between cattle and buffalo and all predicted risk factors were strongly associated with prevalence of subclinical mastitis. The in-vitro antibiotic susceptibility test revealed that cefoxitin was completely resistant, whereas linezolid, ciprofloxacin, and trimethoprim + sulphamethoxazole were sensitive against the MRSA isolates. The relevance of S. aureus to public health, as well as the development of resistance to antibiotics like methicillin, needs ongoing testing of antimicrobial medications against MRSA isolates.