The role of heterotrophic biofilm of water–sediment interface in detoxification processes was tested in abiotic and biotic conditions under laboratory conditions. Three toxicants, a herbicide (Diuron), a fungicide (Dimethomorph) and an insecticide (Chlorpyrifos-ethyl) have been tested in water percolating into columns reproducing hyporheic sediment. The detoxification processes were tested by comparing the water quality after 18 days of percolation with and without heterotrophic biofilm. Tested concentrations were 30 μg.L−1 of Diuron diluted in 0.1% dimethyl sulfoxide (DMSO), 2 μg.L−1 of Dimethomorph and 0.1 μg.L−1 of Chlorpyrifos-ethyl. To characterise the detoxification efficiency of the system, we performed genotoxicity bioassays in amphibian larvae and rotifers and measured the respiration and denitrification of sediments. Although the presence of biofilm increased the production of N-(3,4 dichlorophenyl)-N-(methyl)-urea, a metabolite of diuron, the toxicity did not decrease irrespective of the bioassay. In the presence of biofilm, Dimethomorph concentrations decreased compared with abiotic conditions, from 2 μg.L−1 to 0.4 μg.L−1 after 18 days of percolation. For both Dimethomorph and Chlorpyrifos-ethyl additions, assessment of detoxification level by the biofilm depended on the test used: detoxification effect was found with amphibian larvae bioassay and no detoxification was observed with the rotifer test. Heterotrophic biofilm exerts a major influence in the biochemical transformation of contaminants such as pesticides, suggesting that the interface between running water and sediment plays a role in self-purification of stream reaches.