In this study, twenty five swabs and twenty five biopsies were collected from twenty five patients suffering from chronic osteomyelitis (one biopsy and one swab from each patient) from Al-Yarmouk Teaching Hospital , Surgical Specialist Teaching Hospital and Baghdad Teaching Hospital in Baghdad during the period June 2010 to May 2011 . Results showed that the most common affected gender with chronic osteomyelitis were males with 84% , and the highest incidence were recorded at the age group of 30-39 years distributed between (7) for males (28%) and (3) for females (12%) . Depending on cultural , microscopic examination and biochemical characterization , results of both swab and biopsy specimens showed that 50% of the isolates were Staphylococcus aureus , 26% Enterobacter cloacae , 14% Pseudomonas aeruginosa , 6% Escherichia coli , and 4% Klebseilla sp. Results of the antimicrobial susceptibility test showed wide variation in response to different (11) antibiotic used . All isolates of S. aureus were totally resistance to methicillin , vancomycin , cephalothin and tetracycline followed by clindamycin, gentamicin and penicillin with percentage of resistance of 83% . On the other hand, all S. aureus isolates were completely susceptible to ciprofloxacin , chloramphenicol , novobiocin and rifampicin . A similar result was obtained, when three isolates of Enterobacter cloacae were resistant to ampicilllin, amoxicillin, cephalothin, tetracycline, vancomycin, rifampicin and penicillin , two of them resistant to chloramphenicol and trimethoprim and only one isolate resisted gentamicin . All were susceptible to nalidixic acid and ciprofloxacin . Regarding Pseudomonas aeruginosa and E.coli, results of antimicrobial susceptibility showed that they were susceptible to ciprofloxacin but resistant to all other groups of antibiotic . Klebsiella sp. isolate was susceptible to chloramphenicol and resistant to all other groups of antibiotic. Depending on the antibiotic sensitivity results, four isolates of S. aureus were selected designated as (S1 , S3 , S4 , S7 due to their multiple resistances to antibiotics) to determining the minimum inhibitory concentrations (MIC) of four antibiotics (cephalothin, methicillin, tetracycline, vancomycin) . Results showed that the MICs of methicillin were 320μg/ml for S1 , 80μg/ml for S3, 40μg/ml for S4 and 80μg/ml for S7 . As well as, the MICs of vancomycin were 160μg/ml for S1, 640μg/ml for S3, 80μg/ml for S4 and 40μg/ml for S7 . In cephalothin , 320μg/ml for S1, 80μg/ml for S3, 640μg/ml for S4 and 160μg/ml for S7 . In tetracycline, 320μg/ml , 80μg/ml , 640μg/ml and 40μg/ml for S1 ,S3 , S4 and S7 , respectively . In order to investigate the virulence factors of Staphylococcus osteomyelitis, four S. aureus isolates were found to be able for produce virulence factors like capsule , slime layer , biofilm , hyaluronidase and β- haemolytic of human blood . It was found that all S. aureus isolates were able to produce haemolysin enzyme , capsules, and the capsule stain method. gave more clear and accurate scene than the negative stain method . Also they were able to produce slime layer by using Congo red agar method, it was useful and more specific method to differentiate between producing and non producing isolates . As well as , able to produce biofilm; three of them were highly producers, while , the last revealed moderate production of biofilms . And production of hyaluronidase enzyme by using plate and turbidity reduction methods . In plate method, the diameters of hydrolyzed zones were 7 mm, 6 mm, 18 mm and 27 mm for S1, S3 , S7 and S4 isolates, respectively. Plate method was faster and superior to distinguish between hyaluronidase producing from non producing isolates than turbidity reduction method which showed that four producing isolates caused reduction in the turbidity of medium .