The present paper reports on results obtained from experiments carried out in a laboratory-scale anaerobic packed bed biofilm reactor (APBR), with recirculation of the liquid phase, for continuously biohydrogen production via dark fermentation. The reactor was filled with Kaldnes® biofilm carrier and inoculated with an anaerobic mesophilic sludge from a urban wastewater treatment plant (WWTP). The APBR was operated at a temperature of 37 °C, without pH buffering. The effect of theoretical hydraulic retention time (HRT) from 1 to 5 h on hydrogen yield (HY), hydrogen production rate (HPR), substrate conversion and metabolic pathways was investigated. This study indicates the possibility of enhancing hydrogen production by using APBR with recirculation flow. Among respondents values of HRT the highest average values of HY (2.35 mol H2/mol substrate) and HPR (0.085 L h−1L−1) have been obtained at HRT equal to 2 h.