Bovine mastitis constitutes an economic and serious problem in dairy industry worldwide. This study aimed to determine the phenotypic and molecular characterization of methicillin-resistant Staphylococcus aureus isolated from bovine mastitis in Menoufiya governorate, Egypt. A total of 530 mastitic samples (280 clinical and 250 subclinical) were collected and subjected to bacteriological examination. The result revealed that prevalence rate of clinical and subclinical mastitis was 52.83% and 47.16%, while S. aureus prevalence rate was 28.57% and 24.4% in clinical and subclinical mastitis respectively. Out of 157 staphylococci isolates on MSA medium, 141(89.9%) were identified as S. aureus isolates through biochemical activities and confirmed by amplifying of nuc gene at 279 bp. All S. aureus isolates confirmed by nuc gene (141 isolates) were tested for antibiogram profile against some β-lactams antibiotics (oxacillin, penicillin and amoxicillin/clavulanic acid) that recorded high resistance against these antibiotics. Out of 141 isolates, 128 (90.78%) were confirmed as MRSA strains based on phenotypic resistance to oxacillin and confirmed by molecular detection of the mecA gene at 310 bp. In conclusion, these results showed the significance of continuous surveillance of antibiogram pattern of S. aureus isolates of mastitis origin to design effective control measures for S. aureus mastitis.