Methicillin-resistant Staphylococcus aureus (MRSA) causes a wide range of infections in humans. These infections include pneumonia, abscesses, osteomyelitis, endocarditis, phlebitis, mastitis, and meningitis. This study aimed to investigate MRSA prevalence among patients, outpatients, and health care workers (HCWs) at tertiary university hospitals in Mansoura, Dakahlia. The strains were collected from different sources (puss, blood, eye, urine, HCWs) and PCR was used to detect mecA. The isolates were investigated for their susceptibility to different antibiotic classes and their ability to produce β-lactamases and biofilms. Out of the 200 S. aureus strains, 103 (51.5%) were MRSA. The prevalence of MRSA among inpatients, outpatients, and HCWs was 43.5%, 46.0%, and 61.7%, respectively. MRSA isolates were tested for their susceptibility to 15 antibiotics. More than 50% of the MRSA isolates were resistant to eight antibiotics. Less than 10% of the isolates were resistant to amikacin, ciprofloxacin and lomefloxacin. Alarming resistance levels for vancomycin (23.30%) and linezolid (56.31%) were observed. MRSA strains were multidrug-resistant (MDR) and were resistant to 4-13 antibiotic classes. All MRSA strains were biofilm producers. The strong, moderate and weak biofilm producers were 23.30%, 49.51% and 27.18%, respectively. The percentages of β-lactamase production by inpatients, outpatients, and HCWs were 90%, 95.7%, and 96%, respectively. We may conclude that MRSA isolates are prevalent in outpatients, inpatients, and HCWs in university tertiary hospitals at Mansoura. All MRSA were MDR-resistant and biofilms and most of them were β-lactamase producers. Therefore, infection control procedures must be urgently implemented.