This study proposes an optimization-based strategy to select the best electrical stacking configuration of microbial fuel cells to achieve the highest power output and chemical oxygen demand removal under the lowest operating costs. Three similar wastewater-fed continuous flow microbial fuel cells are electrically connected in four different modes and concentrations of fuel substrate and buffer in anolyte, as main operating cost items, are optimized using two-level factorial design with the dual objective of maximizing power density and minimizing operating costs. In series connection the lowest ratio of operating costs to maximum power $0.048.mW(-1).d(-1) is achieved that is comparable to the ratio of $0.046.mW(-1).d(-1) for an individual unit as control. Optimization reduces operating costs 61% with only 37% reduction in maximum power compared to maximum attainable power. At the optimized concentrations, the lowest operating costs to chemical oxygen demand removal ratio $2.01.COD-1 is observed in series connection. This suggests that the cheapest way to stack microbial fuel cells to gain the highest power output and chemical oxygen demand removal is serial electrical connection. (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.