Little information exists on how various process parameters determine the success of a novel process first proposed in 2005, called the electrochemically assisted microbial cell process. This study attempts to identify the essential process parameters controlling the hydrogen production rates in this electrochemically assisted process. At an applied voltage of 600 mV, microbes in an anode chamber can effectively degrade acetate but produced no hydrogen. The control of neutral pH at the anode chamber was critical to the formation of an active biofilm on the anode surface; consequently, the generated protons and electrons were transferred to the cathode and formed hydrogen gas. Up to 11.6% of electrons produced were transferred to hydrogen in the present mass flow controller system at pH 7.0 and an applied voltage of 540 mV. Competition among other cations through the proton-exchange membrane and the presence of suspended-growth microbial populations correspond to the observed low recovery efficiency of hydrogen from acetate. Cell starvation can result in biofilm deterioration and, thus, system failure.