A dual-chambered microbial fuel cell with aqueous cathode was operated with domestic wastewater to investigate the electrogenic ability of anaerobic bacteria from a municipal wastewater treatment plant. Curves of cell potential vs. current density, power density vs. current density and current at a fixed load of 100 Ω, were obtained daily to monitor the electrochemical evolution of the system as a function of substrate use in several batch cycles. A maximum power density of 1.11 µWcm-2 was obtained after 65 days of continuous operation and a coulombic efficiency of 7% and a chemical oxygen demand removal of 76% were found in the last batch cycle. Anaerobic culture of the bacteria from the anode biofilm resulted in the isolation of two Gram-positive and two Gram-negative bacteria with divergent sugar fermentation capabilities, while analysis of 16S rRNA gene fragments showed three clones from the phyla Firmicutes, δ-Proteobacteria and α-Proteobacteria. Scanning electron imaging analysis revealed an increase in cell diversity and proliferation of methanogenic archaea when changing from synthetic to real wastewater. These results reveal the influence of substrate concentration and presence of methanogenic microorganisms on the production of power in microbial fuel cells, suggesting that future developments could be a contribution for the use of this technology to decontaminate domestic wastewater in small communities.