Sediments are an essential component of aquatic ecosystems in terms of biodiversity and of ecological functioning. They receive direct or indirect inputs from the water column or from the watershed including some toxic and persistent contaminants (e.g. trace metals and PCBs) which can accumulate over time and impact exposed organisms. Among benthic organisms, microbial communities are major players in various key ecological processes such as organic matter recycling, greenhouse gas production and biomass production contributing to benthic food webs. The study of ecotoxicological effects on microbial communities paves the way to assess the impacts of contaminants on both taxonomic and functional microbial biodiversity which support many ecosystem functions and ensure their stability and resilience. Accordingly, microbial ecotoxicology offers prospects to develop new ecosystem quality indicators. However, knowledge about the effects of accumulated contaminants on sediment microbial communities is scarce. In this context, the main aim of this work was to evaluate 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 metagenomic 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 (within 48 hours) and marked effects of Cu alone on the exposed communities. It led to a significant inhibition of microbial functions such as respiration and denitrification as well as beta-glucosidase, leucine aminopeptidase and phosphatase activities, and affected the proportion of functional genes involved in denitrification pathways. 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. They also show that PICT approaches have the potential to be a powerful microbial indicator to assess in situ the ecological quality of metal-contaminated sediments. These results open new perspectives to assess the ecological quality of sediments and confirm the need for developing studies to better understand the ecotoxicological impact of contaminants on natural sediment communities.