Two hydrocarbon-biodegrading bacterial strains, B1 and B2, were isolated from petroleum-contaminated soil collected from Tianjin, China. The strains were identified as Pseudomonas aeruginosa (B1) and Acinetobacter junii (B2). The degradation rate of n-hexadecane by B1 and B2 reached 96% and 78% respectively after 7 days, though the strains employed different mechanisms of degradation. The results showed that B2 was not able to use glucose as carbon source. B1 could produce glycolipid surfactants using glucose as the carbon source, according to the results of blue agar plate analysis and thin layer chromatography (TLC), and the bacterial culture of B1 had a high oil discharge and emulsification activity. Both B1 and B2 could produce biosurfactants with hexadecane as the sole carbon source, but their modes of action were different. The carbon source was found to affect the cell surface hydrophobicity. Cell surface hydrophobicity was poor with glucose as the carbon source, but enhanced when hexadecane was used as the carbon source.