Microbial fuel cell (MFC) is used to remove organic pollutants while generating electricity. Biocathode plays as an efficient electrocatalyst for accelerating the Oxidation Reduction Reaction (ORR) of oxygen in MFC. This study integrated biocathode into a single-chamber microbial fuel cell (BSCMFC) to produce electricity from an organic substrate using aerobic activated sludge to gain more insights into anodic and cathodic biofilms. The maximum power density, current density, chemical oxygen demand (COD) removal, and coulombic efficiency were 0.593 W m−3, 2.6 A m−3, 83 ± 8.4%, and 22 ± 2.5%, respectively. Extracellular polymeric substances (EPS) produced by biofilm from the biocathode were higher than the bioanode. Infrared spectroscopy and Scanning Electron Microscope (SEM) examined confirmed the presence of biofilm by the adhesion on electrodes. The dominant phyla in bioanode were Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, while the dominant phylum in the biocathode was Proteobacteria. Therefore, this study demonstrates the applicable use of BSCMFC for bioelectricity generation and pollution control.