In this research, Electrochemical Impedance Spectroscopy (EIS) is employed as a valuable technique to investigate the microbial fuel cells (MFCs) performance in terms of internal resistances in molecular level. At different operational conditions impedances were evaluated using Nyquist plots and an equivalent circuit fitting method. The results showed that the most important resistance in MFCs is related to substrate oxidation reaction for the generation of electrons that is 15 times higher than other resistances. Contamination of anode chamber in the long run might contribute to the increase of impedance even though lower resistances are expected in the long operation time.

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