Abstract. Background: Staphylococcus aureus is an important causative pathogen. The production of biofilms is an important factor and makes these bacteria resistant to antimicrobial therapy. Objectives: the current study aimed to assess the prevalence of resistance to antibacterial agents and to evaluate the phenotypic and genotypic characterization of biofilm formation among S. aureus strains. Methods: In this study, 50 isolates of Methicillin-resistant S. aureus (MRSA) and Methicillin-Susceptible S. aureus (MSSA) were included. S. aureus was identified by molecular and conventional methods. Antimicrobial resistance was tested with a disc diffusion method. The biofilm formation was performed through Microtiter plate method. Strains were subjected to PCR to determine the presence of nuc, mecA, icaA, icaB, icaC, and icaD gene. Results: Of the total 50 S. aureus isolates, 32(64%) and 18(36%) were MRSA and MSSA respectively. A large number of MRSA and MSSA isolates showed resistance to Penicillin and Azithromycin and a lower number of MRSA and MSSA isolates showed resistance to Amikacin Gentamicin and none of the isolates was resistant to Vancomycin. The MRSA strains had significantly higher resistance against antibiotics than MSSA strains (P = 0.0154). All isolates (MRSA and MSSA) were able to produce biofilm with levels ranging from strong (31.25 %), (16.6%) to moderate (53.12%), (50%) to weak (15.6 %), (33.3% %) respectively. The MRSA strains had significantly higher ability of biofilm formation than MSSA strains (P = 0.0079). The biofilm encoding genes were detected among isolates with different frequencies. The majority of S. aureus isolates 42 (84%), were found to be positive for the icaA. The prevalence rates of the icaB, icaC, and icaD genes were found to be 37 (74%), 40 (80%) and 41 (82%) respectively. Conclusions: The prevalence of biofilm encoding genes, which are associated with multidrug resistance in S. aureus strains, is high. Therefore, identification of epidemiology, molecular characteristics, and biofilm management of S. aureus infection would be helpful.