The performance and anode biofilm formation of two types of microbial fuel cells (MFCs) were studied in this paper. During 175 days of operation of single-chamber microbial fuel cells (SMFCs), the voltage exceeded 300 mV over 74.9% of the running time and was over 500 mV for 49.3% of the time. For dual-chamber microbial fuel cells (DMFCs), the voltage exceeded 300 mV for 54.3% of the running time and was over 450 mV 50.9% of the time. The removal rate of COD reached 83.6% and 80.0% in the SMFCs and DMFCs, respectively. The maximum power outputs were 686 mW m⁻² and 776 mW m⁻² for the two types of MFCs during polarization. In this study, the 454 pyrosequencing technique, a massively parallel sequencing technology, was utilized to survey the microbial community of the anode biofilms in different MFC reactors. A variation existed between the biofilm communities of the two MFC anodes. This was presumably caused by different electron acceptors, which affected the extracellular electron transfer speed.