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Backgrounds and Aim: Staphylococcus aureus biofilms are involved in a multitude of serious chronic infections. Production of biofilms is a defensive-invasive process controlled and regulated by the aap and icaR genes. The expression levels of these genes play an important role in the formation of biofilm. The aim of this study was to investigate the expression of icaR and aap regulatory genes in clinical isolates of S. aureus resistant to methicillin and gentamicin. Materials and Methods: In this analytical study, among 285 samples, we detected 100 isolates of methicillin resistant and 82 isolates of gentamicin resistant S. aureus. Resistant strains were evaluated for the presence of biofilm regulatory genes. The expression levels of regulatory genes were measured by real-time PCR method. We used SPSS software 16 for statistical analysis and also REST 2008 V3 software for analysis of quantitative results. Results: Among 100 methicillin resistant and 82 gentamicin resistant isolates of S. aureus the highest expression levels of icaR and aap genes were detected in the smears obtained from the wounds and catheters. Moreover, a different pattern of gene expression was observed in multidrug resistant strains in comparison to the strains with lower rate of resistance. Also, there was a significant relationship between the presence and activity of regulatory genes and biofilm formation in different samples (p≤0 / 05). Conclusion: Considering the frequency of biofilm producing strains of S. aureus in the smears from the catheters and wounds and also increased gene expression, appropriate therapeutic measures should be considered for methicillin and gentamicin resistant of S. aureus. Keywords: Drug resistance, S. aureus, Virulence factors, Methicillin, Gentamicin, Gene expression. Received: Aug 2, 2017 Accepted: Nov 6, 2017