Sisymbrium irio is a plant used in folk medicine in Asian peoples. In 79 samples collected from hospitals, 71 were infected with Gram-positive bacteria. Performing API 20 samples showed 17(23.94%) were S. pyrogens and 54(76.06%) were S. aureus. In an experiment for antibiotic sensitivity, the samples showed 100% sensitivity to penicillin, cephalexin, cefotaxime, tetracycline, Amoxicillin and methicillin. However, the sensitivity was less in Vancomycin, clindamycin, Rifampin. Moreover, it was resistance to ciprofloxacin. Test tube method used to detectability of pathogenic S. aureus isolates which isolated from the skin of children had impetigo for biofilm formation. The result illustrated the high per cent of S. aureus isolates were able to produce biofilm. 47 (87%) S. aureus isolates produce biofilm with different degree of thickness and only 6 (13%) isolates unable to produce biofilm. The total flavonoids content was determined by spectrophotometer. The ethanol, metabolic and aqueous extract of S. irio as rutin the best standard substance for flavonoids. The best absorbance was methanol extract followed by water then, ethanol extracts. Reductive ability was carried out to know the effect of free radicals. The best extract was methanol followed by ethanol then, water extract.