In the arsenic biogeochemical cycle, microorganisms may affect arsenic toxicity by changing its speciation. Gold mining activities in fluvial systems may cause arsenic pollution, as in Anllóns River (Galicia, NW Spain), where high concentrations in surface sediments (up to 270 mg/kg) were found. A 51 days-long biofilmtranslocation experiment was done in the Anllóns River, moving from a less to a more polluted site, to explore the effect of epilithic biofilm on arsenic retention and speciation in the water-sediment interface. Eutrophic conditions were detected at both sites. There were identified 41 species of diatoms at the end of the experiment, being Cocconeis placentula Ehrenberg var. placentula (55.37 %) and Mayamaea atomus (Kützing) Lange-Bertalot var. atomus (18.34 %) the most abundant species. No significant differences were found between translocated and non-translocated biofilms in diatom diversity indices (S, H and J). However, translocated biofilms accumulated more arsenic and their growth was reduced to half that observed in nontranslocated. Methylated As-species (DMA V) were found in intracellular biofilm compartment, what may suggest a detoxification process by biofilm (methylation). Detection of DMA V in water may indicate that biofilm contributes to arsenic speciation in the interface water-epilithic biofilm. Moreover, arsenate (As V) reduction by biofilm may be confirmed by the high amount of arsenite (As III) detected in its extracellular compartment. Our study provides new arguments for the understanding of the key role of microorganisms in the arsenic biogeochemical cycle in freshwater environments.