The membraneless bioelectrochemical reactor (Ml-BER) is useful for dark hydrogen fermentation. The effect of the electrochemical reaction on microorganisms in the Ml-BER was investigated using glucose as the substrate and compared with organisms in a membraneless non-bioelectrochemical reactor (Ml-NBER) and bioelectrochemical reactor (BER) with a proton exchange membrane. The potentials on the working electrode of the Ml-BER and BER with membrane were regulated to -0.9 V (versus Ag/AgCl) to avoid water electrolysis with a carbon electrode. The Ml-BER showed suppressed methane production (19.8 ± 9.1 mg-C·L(-1)·day(-1)) and increased hydrogen production (12.6 ± 3.1 mg-H·L(-1)·day(-1)) at pHout 6.2 ± 0.1, and the major intermediate was butyrate (24.9 ± 2.4 mM), suggesting efficient hydrogen fermentation. In contrast, the Ml-NBER showed high methane production (239.3 ± 17.9 mg-C·L(-1)·day(-1)) and low hydrogen production (0.2 ± 0.0 mg-H·L(-1)·day(-1)) at pHout 6.3 ± 0.1. In the cathodic chamber of the BER with membrane, methane production was high (276.3 ± 20.4 mg-C·L(-1)·day(-1)) (pHout, 7.2 ± 0.1). In the anodic chamber of the BER with membrane (anode-BER), gas production was low because of high lactate production (43.6 ± 1.7 mM) at pHout 5.0 ± 0.1. Methanogenic archaea were not detected in the Ml-BER and anode-BER. However, Methanosarcina sp. and Methanobacterium sp. were found in Ml-NBER. Prokaryotic copy numbers in the Ml-BER and Ml-NBER were similar, as were the bacterial community structures. Thus, the electrochemical reaction in the Ml-BER affected hydrogenotrophic and acetoclastic methanogens, but not the bacterial community.