Hydrogen is a key intermediate element in microbial electrosynthesis as a mediator of the reduction of carbon dioxide (CO₂) into added value compounds. In the present work we aimed at studying the biological production of hydrogen in biocathodes operated at − 1.0 V vs. Ag/AgCl, using a highly comparable technology and CO₂ as carbon feedstock. Ten bacterial strains were chosen from genera Rhodobacter, Rhodopseudomonas, Rhodocyclus, Desulfovibrio and Sporomusa, all described as hydrogen producing candidates. Monospecific biofilms were formed on carbon cloth cathodes and hydrogen evolution was constantly monitored using a microsensor. Eight over ten bacteria strains showed electroactivity and H₂ production rates increased significantly (two to eightfold) compared to abiotic conditions for two of them (Desulfovibrio paquesii and Desulfovibrio desulfuricans). D. paquesii DSM 16681 exhibited the highest production rate (45.6 ± 18.8 µM min⁻¹) compared to abiotic conditions (5.5 ± 0.6 µM min⁻¹), although specific production rates (per 16S rRNA copy) were similar to those obtained for other strains. This study demonstrated that many microorganisms are suspected to participate in net hydrogen production but inherent differences among strains do occur, which are relevant for future developments of resilient biofilm coated cathodes as a stable hydrogen production platform in microbial electrosynthesis.