The performance of miniaturized microbial fuel cells operating with five different substrates (acetate, lactate, glucose and octanoate) were studied with the aim to identify the reason for its different performance. In all cases, the COD removal rate was about 650 mg COD L-1 d-1. However, the bio-electrochemical performance of the MFC was very different, showing the MFC fed with acetate the best performance: 20 A m-2 as maximum current density, 2 W m-2 of maximum power density, 0.376 V of OCV and 12.6% of CE. In addition, the acetate showed the best bio-electrochemical performance in the polarization curves and cyclic voltammetries. These polarization curves were modelled and the key to explain the better electrical performance of acetate was its lower ohmic losses. When working with acetate, its ohmic losses were one log-unit below those attained by the other substrates. These lower ohmic losses were not associated to the electrolyte conductivity of the fuel but to the lower ohmic loses of the biofilm generated.