The effects of inoculum species, substrate concentration, temperature, and cathodic electron acceptors on electricity production of microbial fuel cells (MFCs) were investigated in terms of start-up time and power output. When inoculated with aeration tank sludge, this MFC outperformed the cell that was inoculated with anaerobic sludge in terms of start-up time and power output. After running for a certain time period, the dominant populations of the two MFCs varied significantly. Within the tested range of substrate concentration (200–1800 mg L−1), the voltage output increased and the time span of the electricity generation lengthened with increasing substrate concentration. As the temperature declined from 35 to 10 °C, the maximum power density reduced from 2.229 to 1.620 W m−3, and anodic polarization resistance correspondingly dropped from 118 to 98 Ω. The voltage output of MFC–Cu2+ was 0.447 V, which is slightly lower than that achieved with MFC–[Fe(CN)6]3− (0.492 V), thereby indicating that MFCs could be used to treat wastewater containing Cu2+ pollutant in the cathode chamber with removal of organics in anode chamber and simultaneous electricity generation.