A laboratory-scale two-chamber microbial fuel cell employing an aerated cathode with no catalyst was inoculated with mixed inoculum and acetate as the carbon source. Electrochemical impedance spectroscopy (EIS) was used to study the behavior of the MFC during initial biofilm (week 1) and maximum power density (week 20). EIS were performed on the anode chamber, biofilm (without anolyte) and anolyte (without biofilm). Nyquist plots of the EIS data were fitted with two equivalent electrical circuits to estimate the contributions of intrinsic resistances to the overall internal MFC impedance at weeks 1 and 20, respectively.