Backgrounds: Staphylococcus aureus is one of the major causes of nosocomial infections. Biofilm formation is an important virulence factor of S. aureus, leading to its high resistance to antibiotics and evasion from host defenses. This study aimed to assess the prevalence and antimicrobial resistance profile of biofilm-producing S. aureus strains and characterize genes involved in biofilm formation. Materials & Methods: A total of 79 S. aureus strains were isolated from 1000 clinical samples and characterized using phenotypic, biochemical, and molecular tests. The biofilm production ability of isolates was examined using the microtiter assay. Moreover, the expression of genes involved in biofilm production (psm A and psm B) was screened using real-time PCR. Finally, antibiotic susceptibility testing was done using the Kirby-Bauer method and interpreted according to the CLSI M100 standard. Findings: Out of 79 S. aureus isolates, 43 (54.4%) isolates were strong biofilm producers, 21 (26.6%) isolates were weak biofilm producers, and 15 (19%) isolates were non-adhesive. The results of real-time PCR showed that 55 (86%), 60 (93.7%), and 46 (58.2%) isolates were positive for psm A, psm B, and both genes, respectively. The results of antibiotic susceptibility testing showed that all the isolates were resistant to two or more antibiotics. Conclusion:The high prevalence of biofilm-forming S. aureus strains in hospital environments could be a major health challenge with serious outcomes for hospitalized patients. Thus, it is necessary to disinfect hospital environments to reduce the risk of infection and spread of these microorganisms.