The study included identification of twenty five Staphylococcus aureus isolates, which were isolated from urine and blood. All these isolates were identified as coagulase and catalase positive, able to ferment mannitol. Tests for the detection of DNase and thermonuclease (TNase) production also gave positive results. The isolates gave similar reactions in API tests in comparison with S. aureus ATCC 25923. Antibiotic sensitivity pattern was demonstrated for all the isolates and multiple resistance of some isolates was detected. The obtained results showed that (62.5 %) of the isolates were resistant to streptomycin and tetracycline, (56.3 %) were resistant to carbenicillin and erythromycin, (45 %) were resistant to methicillin. While, (43.8 %) were resistant to chloramphenicol, (31.3 %) were resistant to cefotaxime, (31%) were resistant to fusidic acid, (25 %) were resistant to neomycin, and (18 %) were resistant to vancomycin. Strain number 24 was chosen and undergone mutation with NTG to isolate temperature sensitive osmotically fragile (TOF) mutants. TOP mutants were successfully isolated by exposing the cell suspension to 200ug /ml of NTG for 75 minutes. The results of characterization of TOF mutants, showed that these mutants were identical to their mother strain morphologically and biochemically. Biotyping of TOF mutants gave the same biotype (B) as the mother strain. Plasmid profile study of TOF mutants using lysozyme was performed. The obtained results showed presence of one plasmid band which might represent a big plasmid and two plasmid bands which might indicate the presence of one plasmid of two forms or to two plasmids.