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Bio-hydrogen (H 2 ) production in a thermophilic anaerobic bioreactor with carbon dioxide (CO 2 ) utilization at the headspace was investigated at different ratio of Biomass substrate to Microorganism (B/M). A 3 mL volume of 80% NaOH (m/v) solution was continuously hung at the headspace of the bioreactors to capture carbon dioxide. The utilization of CO 2 at its headspace, proved to enhance bio H 2 production. The highest Cumulative Biohydrogen Yield (CHY) of 131.81±3.47 mL-H 2 /gVS was measured at the ratio of 8.10 parts of biomass to 0.90 parts of Acclimatized Seed Sludge (ASS), B/M 9, while 4.17 parts of similar biomass to 0.83 parts of ASS (B/M 5) had a biohydrogen production of 90.70±16.67 mL-H 2 /gVS. The B/M of 6.12 parts of biomass to 0.88 parts of ASS (B/M 7) produced a CHY of 84.72±18.35 mL-H 2 /gVS while the control bioreactors without CO 2 utilization (without and with biomass substrate) yielded a 0.06±0.035 mL-H 2 /gVS and a 3.27±0.78 mL-H 2 /gVS respectively. The mechanism of the biofermentation in this anaerobic reaction produced two possible resulting reaction; the acetogenesis of CO 2 with H 2 and the hydrogenotrophic methanogenesis. These reactions consumes hydrogen in the process to produce methane or acids. The presence of 80% (m/v) NaOH solution at the headspace inside the bioreactors, utilizes the CO 2 producing a hydrogen-rich region in space: The highest average H 2 yield of 51.83 mL-H 2 after 49.1 h with B/M 9 without methane was due to carbon dioxide utilization in the bioreactors. A univariate ANOVA and Pair-wise Tukey HSD statistical analysis revealed that the CHY of B/M9 was significantly higher than the other B/Ms. The highest yield, 55.85 mL-H 2 /gVS obtained with the bioreactor of B/M 9 was optimum for H 2 production. The results concluded that H 2 production is also enhanced by CO 2 utilization at the headspace.