Background: Staphylococcus aureus (S.aureus) is one of the gram-positive bacteria that causes a wide range of nosocomial infections. Our present study aimed to investigate genotypic and phenotypic aspects involved in biofilm formation in methicillin-resistant S.aureus strains isolated from hospital-acquired infections at Beni-Suef University Hospital Methodology: A total of 100 patients, 86 S. aureus strains were collected from all departments of Beni Suef University Hospital. The   antibiotic resistance pattern, phenotypes of biofilm formation and ica C gene were studied using Congo Red Agar (CRA) then molecular conformation was done by PCR. Results: We found that 80 out of 86 samples (93%) were mecicillin resistant S. aureus (MRSA). The highest frequency of resistance was found for oxacillin (95.3%), cefoxitin (88.4%), and ceftazidime (82.6%). Phenotypic results showed that 68.8% were high biofilm producers, while 10% and 21.2% were intermediate and low biofilm producers, respectively. From the 63 strong and intermediate biofilm producers isolates, we found that 55 specimens had the icaC gene (87.3%). Conclusion: Our study concluded that adherence ability and biofilm production are important for enhancing virulence factors among isolates of S. aureus strains.