Gold mining activities in fluvial systems may cause arsenic (As) pollution, as is the case at the Anllóns River (Galicia, NW Spain), where high concentrations of arsenate (AsV) in surface sediments (up to 270 mg kg−1) were found. A 51 day-long biofilm-translocation experiment was performed in this river, moving some biofilm-colonized substrata from upstream (less As-polluted) to downstream the mine area (more As-polluted site), to explore the effect of As on benthic biofilms, as well as their role on As retention and speciation in the water-sediment interface. Eutrophic conditions (range: 0.07-0.38 mg L−1 total phosphorus, TP) were detected in water in both sites, while sediments were not considered P-polluted (below 600 mg kg−1). Dimethylarsenate (DMAV) was found intracellularly and in the river water, suggesting a detoxification process by biofilms. Since most As in sediments and water was AsV, the high amount of arsenite (AsIII) detected extracellularly may also confirm AsV reduction by biofilms. Furthermore, translocated biofilms accumulated more As and showed higher potential toxicity (higher As/P ratio). In concordance, their growth was reduced to half that observed in those non-translocated, became less nutritive (less nitrogen content), and with higher bacterial and dead diatom densities. Besides the high As exposure, other environmental conditions such as the higher riparian cover at the more As-polluted site could contribute to those effects. Our study provides new arguments to understand the contribution of microorganisms to the As biogeochemistry in freshwater environments