The aim of this study was to isolate hydrocarbons-degrading bacteria for treatment of oily wastewater from long-standing petroleum-polluted sediments in Bohai Bay, China. Six hydrocarbons-degrading bacteria were screened and identified as Pseudomonas sp. and Bacillus sp. A new approach using a combination of various bacterial species in petroleum biodegradation was proposed and evaluated for its degradation characteristics. Gas chromatography-flame ionization detection (GC-FID) analysis showed that mixed bacterial agents (YJ01) degraded 80.64% of crude oil and 76.30% of crude oil alkanes, exhibiting good biodegradation effect. Besides, after 14 days of culture, the biodegradation assessment markers, pristane and phytane, showed significant degradation rates of 46.75% and 78.23%, respectively. Kinetic analysis indicated that the degradation trends followed a single first-order kinetics model and the degradation half-life (t1/2) of 15 g/L crude oil was significantly shorter (5.48 days). These results indicated that YJ01 could degrade a wider range of hydrocarbons as well as some recalcitrant hydrocarbon components, and can be applied for bioremediation and treatment of oil-contaminated environment.