Biodiesel produced from rapeseed oil was used as a cost-effective and sustainable agent to enhance crude oil biodegradation in sand microcosms. The initial concentration of crude oil and total petroleum hydrocarbon (TPH) was 20,000 and 18,750 mg/kg, respectively. The mass ratio of biodiesel to crude oil was 0 (designated T1), 1:10 (designated T2), 1:4 (designated T3), and 1:2 (designated T4). After 80 days of incubation, the total removals of TPH and PAHs were 68.6 and 61.5% in T1, 78.0 and 67.3% in T2, 86.3 and 76.2% in T3, 72.2 and 57.9% in T4, respectively. Higher amounts of biodiesel reduced TPH biodegradation due to the decreased transfer of substrates caused by dilution effect. The addition of biodiesel stimulated bacterial growth during the initial period but the petroleum hydrocarbon degradation is not always correlated with the density of bacteria in the presence of biodiesel. Dehydrogenase activity (DHA) and polyphenol oxidase (PPO) activity increased greatly after the beginning of incubation. From then on, DHA continuously decreased with time. T3 had the highest DHA and PPO activity from day 30 to the end of the experiment. The lowest toxicity was observed in T3 at day 80, and T3 showed the highest degradation rate constant.