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Total petroleum hydrocarbon (TPH) contamination poses threats to ecological systems and human health. Many studies have reported its negative impacts on soil microbes, but limited information is known about microbial change and response to multiple TPH contamination events. In this study, we investigated TPH contamination level, microbial community structure and functional genes at a multi-contaminated industrial site in Lanzhou, where a benzene spill accident caused the drinking water crisis in 2014. TPHs distribution in soils and groundwater indicated multiple TPH contamination events in history, and identified the spill location where high TPH level (6549 mg kg−1) and high ratio of low-molecular-weight TPHs (>80%) were observed. In contrast, TPH level was moderate (349 mg kg−1) and the proportion of low-molecular-weight TPHs was 44% in soils with a long TPH contamination history. After the spill accident, soil bacterial communities became significant diverse (p = 0.047), but the dominant microbes remained the same as Pseudomonadaceae and Comamonadaceae. The abundance of hydrocarbon-degradation related genes increased by 10–1000 folds at the site where the spill accident occurred in multi-contaminated areas and was significantly related to 2-ring PAHs. Such changes of microbial community and hydrocarbon-degradation related genes together indicated the resilience of soil indigenous microbes toward multiple contamination events. Our results proved the significant change of bacterial community and huge shift of hydrocarbon-degradation related genes after the spill accident (multiple contamination events), and provided a deep insight into microbial response at industrial sites with a long period of contamination history. [Display omitted] • First report on petroleum contamination profiles of Lanzhou benzene exceedance accident. • Multiple contaminated sites were recognized by high concentration of light molecular weight hydrocarbons. • Significant change of microbial community diversity after multiple contamination events. • Increasing abundance of hydrocarbon degradation related genes after multiple contamination events. [ABSTRACT FROM AUTHOR]