Biofilm is a matrix in which a microorganism encases in it and survives environmental stresses. It help the organisms to resist the antibiotics and disinfectant, chronic biofilm associated infection lead to significant increase in morbidity and mortality especially patient with indwelling medical devices. The objective of this research was to analyze the effectiveness of chlorhexidine, sodium hypochlorite and antimicrobial activity of methicillin and vancomycin against biofilm of isolated strains of Staphylococcus aureus isolated from different clinical samples. The results revealed that most biofilm strains were sensitive to vancomycin, some strains were sensitive some were moderate resist and some were resist to methicillin. In comparing different concentrations (0.3%, 0.2%, 0.15% and 0.075%) of chlorhexidine among time interval (1 min, 3 min and 5 min) concentrations showed significant decrease in biofilm formation in association with time; P value (0.001, 0.001, 0.000 and 0.000, respectively). Different concentrations (5%, 4%, 2.5% and 1.25%) of sodium hypochlorite also tested through the same time intervals; concentrations showed significant decrease in biofilm in association with time; P value (0.000, 0.000, 0.000 and 0.000, respectively).