This study was planned to determine the phenotypic, genotypic, and antibiogram characterization of S. aureus recovered from bovine subclinical mastitis in Menoufiya Governorate, Egypt. A total of 140 (28%) milk samples were positive by the California Mastitis Test (CMT) collected from 500 lactating cows. One hundred and five samples were positive on Baird parker agar and Mannitol salt agar which were confirmed by coagulase test and amplification of the nuc gene into 43 (30.7%) S. aureus. Sensitivity test against 9 antibiotics for S. aureus isolates revealed that the highest rate of antibiotics resistance was for penicillin (90.69%), oxacillin (81.39%), chloramphenicol (58.14%) and tetracycline (53.48%), and 33 (76.74%) of the S. aureus identified as MRSA strains and exhibited multidrug-resistant (MDR), while the highest sensitivity for gentamicin (76.76%), both amoxicillin/clavulanic acid and vancomycin (69.77% for each) and ciprofloxacin (62.79%). Furthermore, fifteen isolates were selected for detecting the presence of antibiotic-resistance and virulence genes among S. aureus strains. The mecA was the most prevalent gene (100%) among S. aureus strains followed by blaZ (80%), tetK (66.7%) and ermB (40%) genes with no detection of the vanA gene. Moreover, coa and spa virulence genes were detected in all tested isolated. In conclusion, our results indicate the importance of the regular surveillance of phenotypic and genotypic profiles for S. aureus isolates to ensure effective control measures for bovine mastitis and minimizes the evolution of MDR strains.