While many metals are known to accumulate in sediments, knowledge about the resulting ecotoxicological effects on sediment microbial communities is scarce. The present study aimed at evaluating the structural and functional impact of chronic exposure to environmental concentrations of copper (Cu) and arsenic (As), alone or mixed together, on river sediment microbial communities. Natural uncontaminated surface sediments collected in a French River (Ain) were spiked with As and Cu at a nominal concentration of 40 mg/kg and then were exposed for 21 days in laboratory channels with overlying waters. The response of heterotrophic microbial communities to metals was evaluated both in terms of genetic structure (using ARISA analysis) and functional potential (using exo-enzymatic, metabolic and qPCR analyses). A pollution induced community tolerance (PICT) approach was also performed to assess if the exposure led to an increase in the capacity of microbial communities to tolerate metals. Our results showed rapid and marked effects of Cu alone on the structure and the functions of the exposed communities. Chronic Cu exposure also induced an increase in community tolerance to Cu, as observed by PICT measurement using beta-glucosidase activity. In contrast, the effects of As were mostly undetectable. Under mixture exposure (Cu+As), the effects were similar or higher than those provoked by Cu alone, depending on the measured parameter. Altogether those findings reveal that metals accumulation in sediments can impact exposed microbial communities thus affecting their functional role in aquatic ecosystems.