Total pressure (TP) affects the level of dissolved hydrogen gas in the fermentation medium leading to metabolic shifts in mixed microbial-culture-based systems. In this study, the effect on hydrogen production rate and yield was investigated at different TP of a hydrogen-producing system using a microbial non-sterile culture previously heat-treated. Four continuous stirred-tank reactors (CSTR) were operated in parallel on a mineral salts-molasses medium (21 g-COD. L−1) at 35 °C, pH 5.5 and hydraulic retention time (HRT) of 6 h. The TP was set at 80 kPa (R1), 100 kPa (R2), 120 kPa (R3) and 140 kPa (R4) for which reactor performances were estimated at steady-state conditions. As the increase of TP consequently increased the partial pressure of hydrogen (), the hydrogen production rate (HPR) and yield (HY) were consistently negatively influenced. The highest HPR and HY (406.1 ± 36.8 mL-H2 h−1; 4.51 molH2 mol−1suc eq.) were achieved at low pressure conditions (80 kPa). The composition of the microbial community mainly represented by species from Sporolactobacillus and Clostridium genera, did not change with the increase and/or decrease of the TP, indicating a regulation at cellular but not population level.