Air-cathode single-chamber microbial fuel cells (MFCs) have potential for practical applications in bioenergy production. One of the main challenges is that the coulombic efficiency (CE) is relatively low compared with MFCs that contain a membrane. In this study, unlaminated carbon was used as separator in MFCs with three different configurations to enhance the CE and to increase power by shortening the distance between the anode and cathode. A CE of 28% (1000 Ω load) was obtained in the MFC using the unlaminated carbon as a separator on the cathode side with 2 cm distance between the anode and cathode, which was an increase of 50% compared to a control lacking a separator. The CE was further enhanced to 47% (1000 Ω) using a separator electrode assembly (SEA) configuration, where the separator was sandwiched between the anode and cathode. The power density also was increased by the SEA configuration, and the internal resistance decreased from 64 Ω (2 cm electrodes space) to 41 Ω (SEA). A double SEA MFC (with the pairs of electrodes 2 cm apart) produced 41 W/m3, which is an increase of 140% compared to that with single electrodes spaced 2 cm apart. This study showed that a separator could increase the CE, and that the use of separators allows a smaller distance between the electrodes that can increase reactor performance.