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This study aimed to reveal the effect of total flavonoids and crude extract of ficus religosaon biofilm inhibition and to evaluate the synergistic activity of crude and flavonoids with Gentamicin(CN) as a biofilm antagonist of clinically isolated S. aureus. Crude extraction was accomplished using 80% methanol and flavonoid extraction accomplished using 100% methanol. 130 clinical burn, wound and fractures samples were collected from patients at Medical City Hospital. The Vitik 2 assay identified 60 of them as S. aureus and only 46 isolates were able to produce biofilm when grown in red medium and microtiter plates. Molecular detection was carried out for the availability of genes responsible for biofilm formation in bacterial isolates The bacterial isolates were selected according to their ability to form biofilm and their resistance to antibiotics. The results showed that 24 (80%) isolates possess the icaA gene and 26 (86.6%) isolates possess the icaB for S. aureus. The results showed that the crude extract was more effective than flavonoids in inhibiting biofilm production at a concentration of 512 µg /ml. The results also showed that the mixture of Gentamicin (8 µg /ml) and the crude extract (128 µg /ml) against biofilm formation of S. aureus, where the percentage of biofilm inhibition was (86.6%). The flavonoids with Gentamicin against S. aureus were (128 µg/ml) and (8 µg/ml), respectively, where the inhibition ratio was (79.3%). At low levels, this showed synergistic efficacy against bacterial biofilm formation. These results show that this drug can be used to fight S. aureus biofilms well as as an antibacterial drug in the future.