Scaling-up of microbial fuel cells (MFCs) for practical applications requires compact, multiple-electrode designs. Two possible configurations are a separator electrode assembly (SEA) or closely spaced electrodes (SPA) that lack a separator. It is shown here that the optimal configuration depends on whether the goal is power production or rate of wastewater treatment. SEA MFCs produced a 16% higher maximum power density (328 ± 11 mW m-2) than SPA MFCs (282 ± 29 mW m-2), and higher coulombic efficiencies (SEAs, 9-31%; SPAs, 2-23%) with domestic wastewater. However, treatment was accomplished in only 12 h with the SPA MFC, compared to 36 h with the SEA configuration. Ohmic resistance was not a main factor in performance as this component contributed only 4-7% of the total internal resistance. Transport simulations indicated that hindered oxygen diffusion into the SEA reactor was the primary reason for the increased treatment time. However, a reduction in the overall rate of substrate diffusion also may contribute to the long treatment time with the SEA reactor. These results suggest that SEA designs can more effectively capture energy from wastewater, but SPA configurations will be superior in terms of treatment efficiency due to a greatly reduced time needed for treatment. © 2013 Elsevier B.V. All rights reserved.