MRSA is one of the major pathogens in hospitals and the community, which have the ability to produce biofilm as a virulence factor, the impact of chalcone on biofilm formation, the synergism effect of chalcone and antibiotic in both in vitro and in vivo experiments, the gene expression of virulence genes (srtA, fnbA, fnbB) before and after treatment of it on MRSA biofilm cells in vitro, all these were the prime aims of this study. Chalcone at MBIC (20 μg/ml), significantly reduced the biofilm formation to 21.45% and at sub MBIC (15 μg/ml) to 36.58 %. While, Chalcone at MIC(5 μg/ml) reduced MRSA planktonic cells to 49.61%. Susceptibility of MRSA isolates against eight antibiotics showed that all isolates were sensitive to vancomycin and none of the isolates developed susceptibility to erythromycin. The combinatorial effect of chalcone at 5 μg/ml and vancomycin at MIC of (1 μg/ml) on MRSA planktonic cells was reduced it from 70 to 23.3% , and in combination with erythromycin at 32 μg/ml, was decreased from 53.1% to 22% and the effect of chalcone at sub MBIC (15 μg/ml) when combined with vancomycin was reducing the biofilm formation from 87% to 27.6 and with erythromycin from 55.1% to 23.8%. Combinatorial phenotypic effect of the antibiotics and chalcone (at sub MBIC), in vitro came in line with the result of in vivo experiment and the results showed decrease in the expression of fnbA, fnbB and srtA genes in tested isolates in the presence of chalcone at sub MBIC. In our study, we demonstrated that chalcone exhibited significant effect in biofilm formation of MRSA strains, which can be considered as promising antimicrobial agents that can be used for prevention of MRSA adherence or as adjunct to antibiotics in conventional therapy.