Arsenic (As) is naturally worldwide distributed through the Earth crust, and often associated to pyrite, which is one of the most ubiquitous minerals. Arsenic in a fluvial system can appears after a long-term process of rock weathering, downstream transport and sediment deposition of arsenic-rich minerals. Some human activities, like gold mining, accelerate this process. That is the case of Anllóns river in Galicia (northwest Spain), where it was found high concentrations of As (up to 270mg/kg) in surface and subsurface sediments. This study aimed to explore the effect of the biofilm (epilithic) on As, in a polluted section of the Anllóns river. To achieve it, an biofilm-translocation experiment was done, after 5 weeks of colonization over artificial substrata in a less-polluted river site (Verdes), but with similar physicochemical characteristics than the translocated one (Xavarido). The experiment lasted 51 days, and the evolution of environmental parameters (in sediment and water) as well as biofilm responses (algal biomass, photosynthetic responses, chl a, live diatoms, total bacteria, diatom community, C:N:P, and As accumulation and speciation) were analyzed over time. Arsenic accumulation and speciation were analyzed in 3 compartments: sediment, water (pore and river water) and translocated biofilm (extracellular and intracellular). High concentration of phosphate was detected in sediment and water. Results also showed that, in Xavarido, biofilm growth reached half of that in Verdes. Xavarido’s sediments had much more As (specially AsV) and it was more mobilizable than Verdes’ sediments. Very little amount of As was found in water. Biofilms from Xavarido accumulated more As than from Verdes, and arsenic-methilated species (DMA) were found in the intracellular compartment, showing that it was done a detoxification process by biofilm (methylation). This DMA was also found in river water, suggesting that biofilm contribute to the As speciation in Anllóns river. High amount of AsIII was also detected, especially in extracellular compartment, indicating that biofilm could reduced the AsV to AsIII, that is very toxic for the environment. Our study prove that, in the As geocycle, microorganisms (bacteria and algae) have a key role. We conclude highlighting the importance of increasing the understanding of the effect of biofilms on the arsenic cycle to better understand the fate and effects of arsenic pollution at ecosystem level.