Investigating the impact of human activities on marine coastal ecosystems remains difficult because of the co-occurrence of numerous natural and human-induced gradients. Our aims were (i) to evaluate the links between the chemical environment as a whole and microbial diversity in the benthic compartment, and (ii) to compare the contributions of anthropogenic and natural chemical gradients to microbial diversity shifts. We studied surface sediments from 54 sampling sites in the semi-enclosed Toulon Bay (NW Mediterranean) exposed to high anthropogenic pressure. Previously published chemical data were completed by new measurements, resulting in an in depth geochemical characterization by 29 representative environmental variables. Bacterial and archaeal diversity was assessed by terminal restriction fragment length polymorphism profiling on a selection of samples distributed along chemical gradients. Multivariate statistical analyses explained from 45% to 80% of the spatial variation in microbial diversity, considering only the chemical variables. A selection of trace metals of anthropogenic origin appeared to be strong structural factors for both bacterial and archaeal communities. Bacterial terminal restriction fragment (T-RF) richness correlated strongly with both anthropogenic and natural chemical gradients, whereas archaeal T-RF richness demonstrated fewer links with chemical variables. No significant decrease in diversity was evidenced in relation to chemical contamination, suggesting a high adaptive potential of benthic microbial communities in Toulon Bay.