Abstract: Background and Objective: Staphylococcus aureus is commonly associated with mastitis in dairy herds with potential public health implications. This study was conducted to investigate the existence of S. aureus in mastitic milk and to determine the antimicrobial resistance profiles of the isolated strains as well as the resistance and virulence associated genes. Materials and Methods: Two hundred quarter milk samples were collected from 3 dairy farms at Dakahliya (n = 2) and Damietta (n = 1) Governorates, Egypt from September to December 2016. Conventional culturing and Polymerase Chain Reaction (PCR) assays targeting nuc (thermonuclease) and coa (coagulase) genes were performed. Isolates were tested for its susceptibility against 14 antimicrobial agents using disk diffusion method. All the isolates were screened for the presence of β-lactamases (blaZ, mecA) and virulence associated (pvl and tst) genes by PCR. Results: The S. aureus was detected in 42% (84/200) of the total examined milk samples. Regarding the antibiogram results, S. aureus revealed a high resistance against ampicillin (95.2%) and penicillin (83.3%) and a lower resistance was observed against gentamicin (23.8%), amikacin (16.7%) and ciprofloxacin (14.3%). Multidrug resistances were detected in 83.3% of the isolated S. aureus. Of the 70 penicillin-resistant S. aureus isolates, blaZ gene was identified in 67 (95.7%) isolates. Fifty percent of S. aureus isolates harbored the specific amplicon of mecA gene. Markedly, all mecA positive strains displayed multidrug resistance and were also positive for blaZ gene. The virulence determinants pvl and tst were detected in 7.1 and 11.9% of the isolated S. aureus, respectively. Conclusion: Presence of multidrug resistant and toxin producing S. aureus in dairy farms pose a major risk to public health. Therefore, this study highlighted the importance of developing an efficient control program to inhibit the transmission of S. aureus, particularly multidrug resistant strains to humans.