Staphylococcus aureus represents a serious health hazard on both animals and humans. The main goal of the present work was to compare between S. aureus isolated from animal and human origin. This was achieved through studying the virulence, phenotypic characterization, genotypic testing of mecA gene, analysis the antibiotic resistance profile of the recovered S. aureus isolates. A total number of 165 samples were collected from clinical mastitic cows and sheep, pus from abscesses collected from septic wounds of infected animals, respectively. Moreover, blood, pus swabs from abscesses and septic wounds, and sputum samples were collected from diseased humans from Assiut Governorate, Egypt. The results revealed that incidence of S. aureus isolates recovered from the examined animal samples were 8.33%, 100% and 20%, per, while from human cases (blood, pus and sputum) were 16.67%, 53.49% and 75%, independently. Using Staphaurex kits, the prevalence of coagulase positive S. aureus from animal samples reached 8.33%, 100% and 20%, but from the diseased human cases were 16.67%, 53.49% and 75%, respectively. Antimicrobial Sensitivity Testing of the animal isolateswere resistant to cefoxitin (15.79%), tetracycline (10.53%), clindamycin and erythromycin (10.53%), while the isolates weresensitive to gentamicin (100%), trimethoprim + sulfamethoxazole and vancomycin (94.74% for each). However, S. aureus human isolateswere resistant to cefoxitin (75%) and tetracycline (78.57%) and sensitive to vancomycin (100%), ciprofloxacin (89.29%) and trimethoprim+ sulfamethoxazole (82.14%). Out of 47 identified S. aureus strains, 3 from animal isolates (15.79%) and 21 from human isolates (75%) proved to be methicillin-resistant (MRSA). Furthermore, one animal isolate (5.26%) and 12 human isolates (42.86%) were multi-drug resistant (MDR). S. aureus isolates from animal and humans were subjected to genotypic characterization of mecA gene using PCR. All the animal and human isolates were positive for mecA gene with a percentage of 100%. The results of this study provide that from both animals and human samples; the isolation rate of S. aureus was greater than that of MRSA followed by MDR.