With 110-d incubation experiment in laboratory, the responses of microbial quantity, soil enzymatic activity, and bacterial community structure to different amounts of diesel fuel amendments were studied to reveal whether certain biological and biochemical characteristics could serve as reliable indicators of petroleum hydrocarbons contamination in meadow-brown soil, and use these indicators to evaluate the actual ecological impacts of 50-year petroleum-refining wastewater irrigation on soil function in Shenfu irrigation area. Results showed that amendments of ≤1000 mg/kg diesel fuel stimulated the growth of aerobic heterotrophic bacteria, and increased the activity of soil dehydrogenase, hydrogenperoxidase, polyphenol oxidase and substrate-induced respiration. Soil bacterial diversity decreased slightly during the first 15 d of incubation and recovered to the control level on day 30. The significant decrease of the colony forming units of soil actinomyces and filamentous fungi can be taken as the sensitive biological indicators of petroleum contamination when soil was amended with ≥5000 mg/kg diesel fuel. The sharp decrease in urease activity was recommended as the most sensitive biochemical indicator of heavy diesel fuel contamination. The shifts in community structure to a community documented by Sphingomonadaceae within α-subgroup of Proteobacteria could be served as a sensitive and precise indicator of diesel fuel contamination. Based on the results described in this paper, the soil function in Shenfu irrigation area was disturbed to some extent.