The variability of hydrogeochemical conditions can affect groundwater microbial communities and the natural attenuation of organic chemicals in contaminated aquifers. It is suspected that in situ biodegradation in anoxic plumes of chloroethenes depends on the spatial location of the contaminants and the electron donors and acceptors, as well as the patchiness of bacterial populations capable of reductive dechlorination. However, knowledge about the spatial variability of bacterial communities and in situ biodegradation of chloroethenes in aquifers is limited. Here, we show that changes of the bacterial communities, the distribution of putative dechlorinating bacteria and in situ biodegradation at the border of a chloroethenes plume (Bitterfeld, Germany) are related to local hydrogeochemical conditions. Biotic reductive dechlorination occurred along a 50 m vertical gradient, although significant changes of the hydrogeochemistry and contaminant concentrations, bacterial communities and distribution of putative dechlorinating bacteria (Dehalobacter spp., Desulfitobacterium spp., Dehalococcoides spp., and Geobacter spp.) were observed. The occurrence and variability of in situ biodegradation of chloroethenes were revealed by shifts in the isotope compositions of the chloroethenes along the vertical gradient (δ13C ranging from −14.4‰ to −4.4‰). Our results indicate that habitat characteristics were compartmentalized along the vertical gradient and in situ biodegradation occurred with specific reaction conditions at discrete depth. The polyphasic approach that combined geochemical and biomolecular methods with compound-specific analysis enabled to characterize the spatial variability of hydrochemistry, bacterial communities and in situ biodegradation of chloroethenes in a heterogeneous aquifer.