The bacterial diversity of a chronically oil-polluted retention basin sediment located in the Berre lagoon (Etang-de-Berre, France) was investigated. This study combines chemical and molecular approaches in order to define how the in situ petroleum hydrocarbon contamination level affects the bacterial community structure of a subsurface sediment. Hydrocarbon content analysis clearly revealed a gradient of hydrocarbon contamination in both the water and the sediment following the basin periphery from the pollution input to the lagoon water. The nC17 and pristane concentrations suggested alkane biodegradation in the sediments. These results, combined with those of terminal-restriction fragment length polymorphism analysis of the 16S rRNA genes, indicated that bacterial community structure was obviously associated with the gradient of oil contamination. The analysis of bacterial community composition revealed dominance of bacteria related to the Proteobacteria phylum (Gamma-, Delta-, Alpha-, Epsilon- and Betaproteobacteria), Bacteroidetes and Verrucomicrobium groups and Spirochaetes, Actinobacteria and Cyanobacteria phyla. The adaptation of the bacterial community to oil contamination was not characterized by dominance of known oil-degrading bacteria, because a predominance of populations associated to the sulphur cycle was observed. The input station presented particular bacterial community composition associated with a low oil concentration in the sediment, indicating the adaptation of this community to the oil contamination.