Background: Staphylococcus aureus has the ability to form biofilms, and causes significant mortality and morbidity in the patients with wounds. Objective: Our aim was to study the in vitro biofilm-forming ability of isolated S. aureus. Methodology: One hundred clinical isolates of S. aureus were isolated from 350 pus samples using standard microbiological techniques. Biofilm formation ability of these isolates was detected phenotypically by tissue culture plate (TCP) method and congo red agar (CRA) and genotypically by detection of ica ABCD genes by PCR. Results: The clinical isolates of S. aureus recovered from infected wounds exhibited a high degree of biofilm formation. Biofilm formation was observed in (76%), (74%) and (70%) of S. aureus isolates via TCP method CRA and genotypically, respectively. Conclusion: This study illustrated that PCR method can be adopted as the most suitable and reproducible method for detection of biofilm. CRA is qualitative, simple, inexpensive and easily reproducible and convenient as a screening method. TCP is semiquantitative method and remains a precious tool for in vitro screening of different biomaterial for the adhesive properties. Regular surveillance of biofilm formation by S. aureus leads to the early treatment of the wound infection.