Petroleum hydrocarbons released into the environment can be harmful to higher organisms, but they can be utilized by microorganisms as the sole source of energy for metabolism. To investigate the optimal conditions of diesel oil biodegradation, the Plackett-Burman (PB) design was used for the optimization in the first step, and N source (NaNO₃), P source (KH₂PO₄) and pH were found to be significant factors affecting oil degradation. Then the response surface methodology (RSM) using a central composite design (CCD) was adopted for the augmentation of diesel oil biodegradation and a fitted quadratic model was obtained. The model F-value of 27.25 and the low probability value (<0.0001) indicate that the model is significant and that the concentration of NaNO₃N, KH₂PO₄ and pH had significant effects on oil removal during the study. Three-dimensional response surface plots were constructed by plotting the response (oil degradation efficiency) on the z-axis against any two independent variables, and the optimal biodegradation conditions of diesel oil (original total petroleum hydrocarbons 125 mg/L) were determined as follows: NaNO₃ 0.143 g, KH₂PO₄ 0.022 g and pH 7.4. These results fit quite well with the C, N and P ratio in biological cells. Results from the present study might provide a new method to estimate the optimal nitrogen and phosphorus concentration in advance for oil biodegradation according to the composition of petroleum.