Electrochemical impedance spectroscopy (EIS) is in potential a powerful tool for the in depth analysis of microbial fuels cells (MFCs). To prevent the risk of drawing false conclusions from invalid EIS measurements we investigated the feasibility of this method on an MFC by checking: linearity, causality, stability and finiteness. EIS application under steady state conditions was partly feasible. For further application EIS on MFCs we recommend to: (1) use the constant anode or cathode potential measurement mode with a fast couple at the counter electrode; (2) record the polarization curve and measure at different amplitudes to check the linearity condition; (3) perform preliminary measurements to reveal measurement presets; (4) apply prolonged pretreatment to facilitate the stability criterion; (5) perform duplicate measurements to examine the stability; (6) use a broad frequency range to validate the finiteness criterion; (7) use a statistical based validation check based on the Kramers-Kronig transformation.