The current work aimed to isolate Staphylococcus aureus from milk of cows affected by clinical mastitis and to investigate the antimicrobial susceptibility phenotypes of the isolated strains. One- hundred and fifty milk samples were collected from cows with mastitis exhibiting clinical symptoms. Samples were obtained from small holders in the neighboring villages of Mansoura, Dakahalia governorates, Egypt. the bacteriological examination was carried out and The Suspected isolates were subjected for biochemical examination followed by PCR for species identification. S. aureus were then tested for its antimicrobial susceptibility against 18 antimicrobials commonly used for mastitis treatment in Egypt. S. aureus were recovered in a rate of 34% (51/150) from the total examined mastitic milk samples. Antimicrobial sensitivity indicated that the isolates had high susceptibility for imipenem, marbofloxacin and tetracycline (100%  each), ciprofloxacin, vancomycin and ciprofloxacin (98% each),cefotaxime (94.12%), gentamicin (92.2%), cefotaxime (94.12%), ceftriaxone (84.4%), sulfamethoxazole trimethoprim (88.2%), streptomycin (66.7%), while, the recovered S. aureus isolates showed complete resistance (100%) against ceftiofur and oxacillin. While, erythromycin, penicillin, amoxicillin clavulinic and spiramycin showed 55%, 41.2% 39.2% and 39.2% rate of resistance respectively. Multi-drug resistance (MDR) was detected in 94.11% of the retrieved isolates. S. aureus retrieved in a high frequency from the present study and have demonstrated increased resistance to commonly used antimicrobials which considered a serious problem for humans and animals health. Thus, the obtained data on the resistance traits of S. aureus isolated from samples of cow's milk would enable source attribution, risk assessment, and the development of better treatment strategies.