Anthropogenic activities, such as mining and agriculture, have resulted in many freshwater systems having elevated concentrations of copper. Despite the prevalence of this contamination, and the vital ecological function of prokaryotes, just three studies have investigated prokaryote community responses to copper concentration in freshwater sediments. To address this, the current study investigated these communities in outdoor mesocosms spiked with varying copper concentrations. We profiled the prokaryotic communities at the taxonomic level, using next-generation high-throughput sequencing techniques, as well as their function, using baiting with leaf analogues, and Biolog Ecoplates for community-level physiological profiling. Sediments containing just 46 mg kg−1 of copper, had distinctly different microbial communities compared with controls, as determined by both DNA and RNA 16S ribosomal RNA gene (rRNA) profiling. In addition to this, sediment communities displayed a greatly reduced utilisation of carbon substrates under elevated copper, while the communities recruited onto leaf analogues were also disparate from those of control ponds. Given the vital role of prokaryotes in ecosystem processes, including carbon cycling, these changes are potentially of great ecological relevance, and are seen to occur well below the ‘low risk’ sediment quality guideline values (SQGV) used by regulatory bodies internationally.