Microbial-driven organic matter (OM) degradation is a cornerstone of benthic community functioning, but little is known about the relation between OM and community composition. Here we use Rhône prodelta sediments to test the hypothesis that OM quality and source are fundamental structuring factors for bacterial communities in benthic environments. Sampling was performed on four occasions corresponding to contrasting river-flow regimes, and bacterial communities from seven different depths were analyzed by pyrosequencing of 16S rRNA gene amplicons. The sediment matrix was characterized using over 20 environmental variables including bulk parameters (for example, total nitrogen, carbon, OM, porosity and particle size), as well as parameters describing the OM quality and source (for example, pigments, total lipids and amino acids and δ13C), and molecular-level biomarkers like fatty acids. Our results show that the variance of the microbial community was best explained by δ13C values, indicative of the OM source, and the proportion of saturated or polyunsaturated fatty acids, describing OM lability. These parameters were traced back to seasonal differences in the river flow, delivering OM of different quality and origin, and were directly associated with several frequent bacterial operational taxonomic units. However, the contextual parameters, which explained at most 17% of the variance, were not always the key for understanding the community assembly. Co-occurrence and phylogenetic diversity analysis indicated that bacteria–bacteria interactions were also significant. In conclusion, the drivers structuring the microbial community changed with time but remain closely linked with the river OM input.