Microbial biocathodes allow converting and storing electricity produced from renewable sources in chemical fuels (e.g., H2) and are, therefore, attracting considerable attention as alternative catalysts to more expensive and less available noble metals (notably Pt). Microbial biocathodes for H2 production rely on the ability of hydrogenase-possessing microorganisms to catalyze proton reduction, with a solid electrode serving as direct electron donor. This study provides new chemical and electrochemical data on the bioelectrocatalytic activity of Desulfovibrio species. A combination of chronoamperometry, cyclic voltammetry, and impedance spectroscopy tests were used to assess the performance of the H2-producing microbial biocathode and to shed light on the involved electron transfer mechanisms. Cells attached onto a graphite electrode were found to catalyze H2 production for cathode potentials more reducing than −900 mV vs. standard hydrogen electrode. The highest obtained H2 production was 8 mmol L−1 per day, with a Coulombic efficiency close to 100 %. The electrochemical performance of the biocathode changed over time probably due to the occurrence of enzyme activation processes induced by extended electrode polarization. Remarkably, H2 (at least up to 20 % v/v) was not found to significantly inhibit its own production.