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Background and Objectives: Staphylococcus aureus is a common cause of nosocomial infections. The ability of S. aureus to form biofilm and acquire antimicrobial resistance has made this organism a major health problem. In this study, we investigate the biofilm-forming ability of S. aureus isolates from clinical samples. Methods: Sixty S. aureus isolates from clinical specimens were collected from the 5th Azar Hospital of Gorgan (Iran) in 2018. The isolates were identified using conventional methods including Gram staining and biochemical tests (catalase and coagulase). Biofilm formation by S. aureus isolates was evaluated using a microplate-based method. Results: Of 60 S. aureus isolates, 47 (78.3%) strains were identified as biofilm-forming and 13 (21.7%) strains were non-biofilm-forming. Conclusion: The high prevalence of biofilm-producing S. aureus isolates in the 5th Azar hospital of Gorgan could pose a major health challenge with serious consequences for hospitalized patients. Therefore, it is crucial to disinfect and sterilize hospital surfaces and equipment effectively to minimize the risk of contamination and spread of bacteria in the hospital settings. Keywords: Biofilms, Staphylococcus aureus, sample.