The objective of this study was to assess the influence of the hydraulic retention time (HRT) and the pH on the fermentative hydrogen production from a food industry waste (FIW), in a continuous stirred tank bioreactor. Thus, hydrogen production was investigated for HRTs of 12, 8, 6 and 4 h and the results showed a long and stable reactor operation with high hydrogen content in the gas phase and high hydrogen production rates. The optimal HRT was found to be in the range between 6 and 12 h, corresponding to hydrogen yields of 96.27 ± 3.36 and 101.75 ± 3.71 L H2/ kg FIW for 12 and 6 h, respectively. In the sequel, the effect of the pH (in the range 5 – 5.9) was investigated, by changing the buffer solution (BS) components, while the HRT value was maintained at 12 h. As anticipated, the results showed that the operating pH had a significant influence on hydrogen production rates and yields. Characterization of the microbial community was performed at various pH values, giving thus a deeper insight to the well-established hydrogen production process from FIW, since the possible biochemical pathways followed by the microbial consortium, under different operational conditions were elucidated.