The influence of concentration of distillery wastewaters, concentration of inoculum and pH value on hydrogen generation in batch dark fermentation process was studied. Anaerobic digested sludge from municipal purification unit was applied as the source of bacteria mixture. The best specific yield was obtained in system containing 10% v/v of inoculum and 20% v/v of the waste (S0/X0 = 2.8), whereas the maximum amount of hydrogen and the highest rate of reaction was achieved in system containing 25% v/v inoculum and 40% v/v of waste (S0/X0 = 2.2). The content of generated hydrogen in biogas was always higher than 62%. Maximum amount of generated hydrogen was 1 l H2/l medium and the rate was 0.12 l/l/h. Liquid metabolites of hydrogen generation process were mainly acetic and butyric acids. Ethanol and propionic acid were in traces. The ratio of HBu/HAc in medium influenced the yield of generated hydrogen. •Application of distillery wastewater in microbiological hydrogen generation.•Optimization of the reaction conditions.•Determination of major non-gaseous metabolites (for future use in hybrid systems).