The electron recovery in microbial fuel cells (MFCs) is decreased by processes like methanogenesis, bacterial growth, and the accumulation of intermediates. Using a suite of analytical techniques, including electrochemical monitoring, chemical analysis, microsensor analysis, and Titration and Off-Gas Analysis (TOGA), this study aimed to (a) identify and quantify the electron losses occurring at the anode and the cathode of a MFC removing acetate and nitrate (NO3-), respectively, and (b) to investigate the impact of the operational characteristics of the cathode on the denitrification process. Our results show that methane (CH4) production and estimated biomass formation at the anode and nitrous oxide (N2O) accumulation at the cathode were responsible for the reduction of Coulombic efficiency (epsilon) during continuous feeding conditions. At the anode, up to 40.1% of the acetate consumed was released as methane at closed circuit. At the cathode, N2O accumulation represented instead the main loss accounting for up to 10.0 +/- 2.1% of the oxidation capacity of the electron acceptor provided as NO3-. Batch experiments at controlled potentials and currents revealed that for a given current the fraction of electron transferred and released as N2O is significantly reduced by low cathodic potentials.