Sixty three isolates belonged to Bacillus spp. were isolated from 18 soil samples (uncontaminated and contaminated with hydrocarbons and fuels) collected from different locations in Baghdad and Basra governorates. All the isolates (63) were primarily screened for their ability in biosurfactant production depending on blood hemolysis activity at blood agar base, which referred that 34 isolates of the total (63 isolate) were biosurfactant producer. Biosurfactant production ability of the producer isolates was evaluated by surface tension (mN m-1) measurement of cell-free broth after cultivation in mineral salt medium supplemented with sucrose as sole carbon source. Identification tests showed the best producer isolate was Bacillus cereus HI-2. Optimum conditions for biosurfactant production from B. cereus HI-2 were determined. Results indicated that maximum biosurfactant production from this isolate was achieved by using soybean oil (4% v/v), ammonium nitrate (4 g/l), and potassium dihydrogen phosphate (0.5 g/l), at pH 7, 35°C, 180 rpm of shaking, for 72 hrs. Cell-free broth (crude biosurfactant) was collected and purified by acid precipitation method, and the biosurfactant yield was 0.453 gram per liter of culture medium under the optimum conditions. Depending on the FTIR spectroscopy and some biochemical tests (Molisch's and Biuret tests) chemical nature of biosurfactant was characterized. Results elucidated that it was potentially a lipopeptide compound.