Back ground: S. aureus especially methicillin and multi drug resistant S. aureus (MRSA and MDRSA) has the tendency to form biofilm. Our aim was to study the in vitro biofilm-forming ability of S. aureus isolates and to examine the relationship between biofilm formation and antibiotic resistance pattern. Methods: One hundred and forty one clinical isolates of S. aureus were isolated from wound pus and tracheal aspirate samples. Biofilm formation of these isolates was detected by Congo red (CRA) method and micro-titer plate (MTP) methods and confirmed by PCR through detection of biofilm genes. Results: The percentage of biofilm-producing isolates was found to be 81.6% and 58.2% for MTP and CRA methods respectively. The majority of MRSAisolates were positive for biofilm genes. Conclusions: Biofilm-producing isolates exhibit high tendency to develop multidrug resistance and methicillin resistance.