The hyporheic zone, i.e. the water-saturated sediment beneath and alongside the riverbed, is exposed to multiple stressors. Agricultural-watershed rivers are frequently exposed to two concomitant stressors: clogging and copper contamination. However, one stressor exposure can increase sensitivity to a second stressor. The aim of this study was to experimentally test the cumulative effects of these two stressors on copper distribution and structural and functional microbial communities responses in the hyporheic zone. A slow filtration column experiment was conducted to compare the effects of 3 treatments of increasing complexity: 'Reference', 'Copper-contaminated' (dissolved copper added at 191 mu g L-1), and 'Clogging+Copper' (dissolved copper + addition of 2 cm of fine sediment). Microbial community structure and activities were studied at 4 column sediment depths. The results showed that clogging did not modify the distribution of copper, which remained fixed in the first few centimetres. In the first few centimetres, clogging had a stimulating effect on microbial activities whereas copper had limited effects mainly on leucine aminopeptidase activity and microbial community tolerance to copper. The subsurface zone thus hosts significant different microbial communities from the communities in the deeper zones that were protected from surface stressors. This experiment confirms the valuable filtering role played by the hyporheic zone and shows that microbial responses are strongly correlated to microhabitat-scale physicochemical conditions in sediment.