Abstract The production of bio-hydrogen and bio-electricity from biological processes has become a relevant scientific subject in recent years, given that renewable energy can be obtained from organic substrate. Dark fermentation (DF), and microbial electrolysis cells (MECs) have been used to produce bio-hydrogen. In this study, a novel integrated single-stage DF-Microbial fuel cells (MFCs) system was developed to generate bio-hydrogen and electricity simultaneously from wastewater treatment. A proton-exchange membrane fuel cell (PEMFC) was used to produce electricity from the bio-hydrogen gas generated by the integrated system. The effect of hydraulic retention time (HRT) on the bio-hydrogen production and electricity generation was examined. The maximum volumetric bio-hydrogen production rate (VHPR) was 0.44 L H2/L.d (0.66 L H2/g CODremoved), simultaneously obtaining an electricity production of 530 mV (100 mW/m2) at a HRT of 8 d. According to the 16S rRNA gene-based analysis, the microorganisms identified on the anode of the integrated system were: Chryseobacterium, Azotobacter, Bacillus, Enterococcus, Citrobacter, and Methanobacterium. The PEMFC employed to generate voltage using the bio-hydrogen generated from the integrated system was able to produce a maximum voltage of 459 mV (367 mW), with a maximum cell efficiency of 44% (fuel usage of 1.5 × 10−5 mol/h).