The aim of this study is to investigate the biological response of periphyton communities chronically exposed to urban contaminants. Periphyton was grown on three sites on the Seine river (France) along a metallic pollution gradient. Periphyton was collected at days 28, 35 and 42 and the impact of metal exposure was assessed in terms of biomass (Dry Weight, Ash-Free Dry Weight, chlorophyll a), metal accumulation within the biofilms (total and intracellular metal) and tolerance acquisition using a previously developed short-term toxicity test based on the bacterial β-glucosidase activity. In order to reliably compare metal tolerance among biofilms, the EC50 were normalized by the corresponding total suspended solids concentration of the biofilm suspensions used for the test. Metals were tested separately: Cu (day 28), Cd and Zn (day 35), Ni and Pb (day 42). For all the metals tested, the normalized EC50 increased from site 1 to site 3 and reflected the metallic pollution gradient measured in the river water collected at the three sites. Depending on the metal, it was not always possible to inhibit completely the β-glucosidase activity at the contaminated sites (sites 2 and 3), a phenomenon already observed with metal-exposed biofilms in microcosm experiments. This study shows that chronic in situ exposure to environmental concentrations of metals has a significant impact on natural biofilms.