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In this study, twenty two pure bacterial strains isolates were isolated that can use crude oil as a carbon source. They occured at different frequency ratios for each of oil-contaminated soil samples, the produced water, and the crude oil of the Al-Rafidiyah oil field (Basrah south of Iraq). Bacterial isolates were characterized and identified based on phenotypic properties and molecular techniques. Fourteen strains, belonging to three genera of Bacillus sp., Lysinibacillus sp. and Enterobacter sp., were isolated from oil-contaminated soil and nine strains, belonging to Bacillus sp., Lysinibacillus sp., Enterobacter sp. and Brevibacillus sp., were isolated from produced water. Five strains, belonging to two genera, Bacillus sp. and Pseudomonas sp., were isolated from crude oil. New twelve strains were recorded as new strains and deposited in GenBank include Bacillus cereus strain ASWISA1, Bacillus thuringiensis strain ASWISA2, Bacillus sonorensis strain ASWISA3, Bacillus cereus strain ASWISA4, Bacillus subtilis strain ASWISA5, Pseudomonas stutzeri strain ASWISA6, Bacillus subtilis strain ASWISA7, Bacillus cereus strain ASWISA8, Bacillus paramycoides strain ASWISA9, Bacillus paramycoides strain ASWISA10, Brevibacillus brevis strain ASWISA11, and Enterobacter cloacae strain ASWISA12.