The application of a biocathode in a microbial fuel cell (MFC) could be an alternative for the abiotic cathode MFCs that use noble metal catalysts and/or artificial mediators. An open biocathode MFC with oxygen reduction was investigated in this study and the roles of microbes in the cathode compartment were characterized. After 50-days operation, the MFC became stable and the power density of the MFC reached 2.55 W/m3 at an influent flowrate of 0.20 mL/min. The concentration of chemical oxygen demand (COD) was significantly reduced from 372 mg/L (in the influent) to 22 mg/L (in the final effluent) at an influent flowrate of 0.20 mL/min. Microbial community analysis demonstrated that four major groups of the clones were identified, where 28 clone types were derived from the cathode microorganisms, which included proteobacteria, Firmicutes, Bacteroidetes and unclassified bacteria. Among these phylatypes, Deltaproteobacteria was the most abundant division with 25.0% of total clones, which plays important roles in the cathodic electron transfer process. The presence of symmetric peaks could be detected in the effluent of the cathode compartment, which confirmed that the possible electron mediators were excreted by cathodic bacteria involved in the electron transfer process.