This study explored the potential of eutrophic river sediments to attenuate the infiltration of chlorinated aliphatic hydrocarbon (CAH)-polluted groundwater discharging into the Zenne River near Brussels, Belgium. Active biotic reductive dechlorination of CAHs in the riverbed was suggested by a high dechlorination activity in batch- and column biodegradation tests performed with sediment samples, and by the detection of dechlorination products in sediment pore water. Halorespiring Dehalococcoides spp. were present in large numbers in the riverbed as shown by quantification of their 16S rRNA and reductive dehalogenase genes. By using DGGE-fingerprint analysis of relevant nucleic acid markers, it was shown that the Zenne River sediments were inhabited by a metabolically diverse bacterial community. A large diversity of sulfate-reducing bacteria, Geobacteraceae and methanogens, which potentially compete with halorespiring bacteria for electron resources, was identified. The high organic carbon level in the top of the riverbed, originating from organic matter deposition from the eutrophic surface water, resulted in a homogeneous microbial community structure that differed from the microbial community structure of the sediment underneath this layer. Monitoring of CAH concentrations and stable isotope ratios of the CAHs (δ13C) and the water (δ2H and δ18O), allowed to identify different biotic and abiotic CAH attenuation processes and to delineate their spatial distribution in the riverbed. Reductive dechlorination of the CAHs was the most widespread attenuation process, followed by dilution by unpolluted groundwater discharge and by surface water-mixing. During a 21-month period, the extent of reductive dechlorination ranged from 27 to 89% and differed spatially but was remarkably stable over time, whereas the extent of abiotic CAH attenuation ranged from 6 to 94%, showed large temporal variations, and was often the main process contributing to the reduction of CAH discharge into the river. Although CAHs were never detected in the surface water, CAHs were not completely removed from the discharging groundwater at specific locations in the riverbed with high groundwater influx rates. Therefore, it was concluded that an increase in the extent of biotransformation in the riverbed is needed for acceptance of the Zenne biobarrier as a viable remedial option for attenuation of discharging CAH-polluted groundwater.