Polarization, solution-separator, charge transfer, and diffusion resistances of clean and used separator electrode assemblies were examined in microbial fuel cells using current-voltage curves and electrochemical impedance spectroscopy (EIS). Current-voltage curves showed the total resistance was reduced at low cathode potentials. EIS results revealed that at a set cathode potential of 0.3 V diffusion resistance was predominant, and it substantially increased when adding separators. However, at a lower cathode potential of 0.1 V all resistances showed only slight differences with and without separators. Used separator electrode assemblies with biofilms had increased charge transfer and diffusion resistances (0.1 V) when one separator was used; however, charge transfer resistance increased, and diffusion resistance did not appreciably change with four separators. Adding a plastic mesh to compress the separators improved maximum power densities. These results show the importance of pressing separators against the cathode, and the adverse impacts of biofilm formation on electrochemical performance. © 2012 Elsevier Ltd. All Rights Reserved.