Biodegradation is a cost-effective process commonly used to eliminate many xenobiotic hydrocarbons such as diesel oils. However, their hydrophobic character reduces the biodegradation efficiency. In order to overcome this hurdle, kurstakins isolated from Bacillus thuringiensis strain 7SA were used as emulsifying agents. The influence of kurstakin molecules on diesel oil degradation by Acinetobacter haemolyticus strain 2SA was evaluated in the presence and absence of the aforementioned lipopeptide. The degradation rates and gene expressions of alkane hydroxylases were evaluated at days 4, 10, 14 and 21. Results showed that kurstakin molecules increased the hydrophobicity of 2SA. Moreover, diesel oil degradation activities were higher in the presence of kurstakin with 29%, 35%, 29% and 23% improvement at 4th, 10th, 14th and 21st day respectively. Statistical analysis indicated that the difference between the degradation rates in the presence and absence of kurstakin was significant with p = 0.03. The detection of three different hydroxylase genes namely alkB, almA and cyp153 in 2SA genome, might have allowed more efficient degradability of alkanes. According to the real-time PCR results, cyp153 was the most induced gene during diesel oil degradation in the presence and absence of kurstakin. Yet, the three genes demonstrated higher levels of expression in the presence of kurstakin when compared to its absence. This study showed that kurstakins enhance the diesel oil biodegradation rate by increasing the hydrophobicity of 2SA. In addition to their anti-fungal activities, kurstakins can be used as biosurfactant to increase biodegradation of diesel oil.