Hydrogen (H2) is one of the most promising renewable energy sources, anaerobic bacterial H2 fermentation is considered as one of the most environmentally sustainable alternatives to meet the potential fossil fuel demand. Bio-H2 is the cleanest and most effective source of energy provided by the dark fermentation utilizing organic substrates and different wastewaters. In this study, the bio-H2 production was achieved by using the bacteria Acinetobacter junii-AH4. Further, optimization was carried out at different pH (5.0–8.0) in the presence of wastewaters as substrates (Rice mill wastewater (RMWW), Food wastewater (FWW) and Sugar wastewater (SWW). In this way, the optimized experiments excelled with the maximum cumulative H2 production of 566.44 ± 3.5 mL/L (100% FWW at pH 7.5) in the presence of Acinetobacter junii-AH4. To achieve this, a bioreactor (3 L) was employed for the effective production of H2 and Acinetobacter junii-AH4 has shown the highest cumulative H2 of 613.2 ± 3.0 mL/L, HPR of 8.5 ± 0.4 mL/L/h, HY of 1.8 ± 0.09 mol H2/mol glucose. Altogether, the present study showed a COD removal efficiency of 79.9 ± 3.5% by utilizing 100% food wastewater at pH 7.5. The modeled data established a batch fermentation system for sustainable H2 production. This study has aided to achieve an ecofriendly approach using specific wastewaters for the production of bio-H2. •Acinetobacter junii-AH4 can be used for biohydrogen producing potential bacteria.•Two factors affecting biohydrogen production of Acinetobacter junii-AH4 were optimized.•In this study 3L bioreactor was employed for effective hydrogen production.•Maximum hydrogen yield of 1.8 ± 0.09 mol H2/mol glucose by utilizing 100% FWW at pH 7.5.