For practical implementation of microbial fuel cell, increasing power generation is important because it is closely related with energy production rate and wastewater treatability. However, it is not known which relative arrangement of anode and cathode gives the best performance, and it is necessary to know the electrochemical reference point of the brush anode for this. Five different brush-anode configurations were tested in a single-chambered cubic microbial fuel cell. By merely changing a brush anode configuration, power and current densities were increased by 20% and 30%, respectively. The horizontally-positioned anode configuration (H1) with the closest anode-cathode distance produced the highest power and current. EIS showed that anode impedance and full-cell impedance were decreased by 60% and 49%, respectively. Distance between the brush anode and the cathode appreciably affected microbial fuel cell performance and internal resistance of an anode and a full cell. Coulombic efficiency and energy efficiency were not significantly affected by the anode-cathode distance, but the horizontal type cells showed relatively higher coulombic efficiency and energy efficiency, and COD removal rate and shorter batch time. The center of a titanium current collector and the center of carbon fibers of a brush-anode were found to be statistically-significant reference points for microbial fuel cell electrochemistry.