Staphylococcus species has the tendency to form biofilms, and causes significant mortality and morbidity in the patients. Aim: Our study was aimed to determine the prevalence of biofilm production in Staphylococcus aureus isolates from various clinical samples and itsantibiotic sensitivity pattern in the tertiary care hospital. Methods: The study included 150 staphylococcal isolates. Biofilm detection in staphylococci was performed using tissue culture plate (TCP). Results: The TCP method detected total positive biofilm production in 96 (64%) staphylococcal isolates. Out of these strong positive was 52 (54.16%), among there 30 were S. aureus and 22 were Coagulase negative Staphylococcus and moderate positive were 44 (45.83%) 31 were S. aureus and 13 were Coagulase negative Staphylococcus. They were resistant to most antibiotics except vancomycin and linezolid. Conclusions: The clinical isolates of Staphylococcus aureus exhibit a high degree of biofilm formation. Higher rate of antimicrobial resistance is demonstrated by biofilm producers than non-producers. Therefore, we recommend regular surveillance of biofilm formation in S. aureus and their antimicrobial resistance profiles