Hydrogen represents a promising clean fuel for future applications. The biocathode of a two-chambered microbial electrolysis cell (biotic MEC) was studied and compared with an abiotic cathode (abiotic MEC) in order to assess the influence of naturally selected microorganisms for hydrogen production in a wide range of cathode potentials (from -400 to -1800 mV vs SHE). Hydrogen production in both MECs increased when cathode potential was decreased. Microorganisms present in the biotic MEC were identified as Hoeflea sp. and Aquiflexum sp. Supplied energy was utilized more efficiently in the biotic MEC than in the abiotic, obtaining higher hydrogen production respect to energy consumption. At -1000 mV biotic MEC produced 0.89 ± 0.10 m3 H2 d-1 m-3NCC (Net Cathodic Compartment) at a minimum operational cost of 3.2 USD kg-1 H2. This cost is lower than the estimated market value for hydrogen (6 USD kg-1 H2).