Background: Staphylococcus aureus has long been recognized as a major pathogen of urinary tract and hospital acquired infections. Over the last decade antibiotic resistance and biofilm formation potential by pathogens is a major challenge. In this study we screened S. aureus clinical isolates by antimicrobial susceptibilty and biofilm assays to determine biofilm capability of vancomycin and ciprofloxacin resistant isolates. Methods: Six clinical isolates of S. aureus were characterized by biochemical tests and further antibiotic susceptibility of vancomycin and ciprofloxacin were tested against S. aureus clinical isolates by disc diffusion method. Biofilm formation capability of these isolates were performed by microtiter plate, coverslip congored agar and tube assays. Results and disscusion:In this study we found that two isolates were resistant to ciprofloxacin and three isolates were resistant to vancomycin, on the basis of CLSI guidelines. Ciprofloxacin resistant isolates showed moderate biofilm formation while vancomycin resistant showed strong biofilm formation. Conclusion:We have concluded that ciprofloxacin and vancomycin resistant clinical isolates were showed differences in biofilm formation. Staphylococcal isolates having biofilm propensity exhibit more resistance to antibiotics, hence are difficult to treat.