Effects of petroleum contamination on bacterial diversities and enzymatic activities in paddy soils were investigated in the Shenfu irrigation area, the largest area irrigated by oil-containing wastewater for more than 50 yr in northeastern China. Bacterial diversities were determined by conventional colony morphology typing techniques and 16S rDNA polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). Dehydrogenase, hydrogen peroxidase, polyphenol oxidase, urease, and substrate-induced respiration (SIR) were measured to evaluate the effects of petroleum-containing wastewater irrigation on soil biochemical characteristics. Results showed that paddy soil total petroleum hydrocarbon (TPH) concentration in the irrigation area varied from 277.11 to 5213.37 mg kg(-1) dry soil. Soil TPH concentration declined along the gradient of the irrigation channel from up- to downstream. At the current pollution level, the paddy soil TPH concentration was positively correlated with the colony forming units (CFU) of aerobic heterotrophic bacteria (AHB) (r = 0.928, p < 0.001) and the genetic diversity based on DGGE profiles (r = 0.655, p < 0.05). The bacterial diversities in the soils based on colony morphotypes of AHB also increased with TPH concentration (r = 0.598), but not significant statistically (p = 0.052). Analysis of soil enzyme activities indicated a significant positive correlation between soil TPH concentration and activities of dehydrogenases (r = 0.974, p < 0.001), hydrogen peroxidases (r = 0.957, p < 0.001), polyphenol oxidases (r = 0.886, p < 0.001), and SIR (r = 0.916, p < 0.001). On the contrary, the urease activity showed a negative correlation with paddy soil TPH concentration (r = -0.814, p = 0.002), and could be used as a sensitive indicator of petroleum contamination.