Introduction: Biofilm is a highly resistant stage of microorganisms in which they exhibit high resistance towards antimicrobial agents and it also increases their virulence power. Staphylococcus aureus is a Gram positive cocci responsible for many serious infections including community acquired and hospital acquired infection. The severity of these infection increases with biofilm production. So, this study is focused on detection of biofilm production by Staphylococcus aureus and comparison of antimicrobial susceptibility pattern of biofilm producing and non-biofilm producing isolates.Material & Method: This study was done on all clinical samples received in m Department of microbiology, National Institute of Medical Sciences and Research, Jaipur over a period of 5 months from August 2019 to December 2019. Samples were received and processed as per the standard guidelines and antimicrobial susceptibility testing was done according to the standard CLSI guidelines. Biofilm production was detected by Tube method.Result: In this study 47 (48.53%) isolates were identified as Staphylococcus aureus and biofilm production was detected in 23 (48.94%) isolates by Tube method. Among the biofilm producers the sensitivity of first line drugs was very poor. Only Vancomycin, Linezolid and Teicoplanin drugs were effective against biofilm producing isolates.Conclusion: Current study showed the comparison between antimicrobial susceptibility pattern of biofilm producing and non-biofilm producing Staphylococcus aureus and it will help the clinicians in treating such patients and thus it will be helpful in reducing the morbidity rate.