Abstract: The present research focused on fermentative production of hydrogen by anaerobic biofilms from a pretreated mixed microflora in a continuous fixed bed-reactor. Microbial mixed culture was thermally (100°C, by 30 min) and acidic (pH 3.0, 24 h) pretreated. Metagenomic analysis of the pretreated microbial cultured was conducted. Microbial cultured community was mainly formed by lactic acid bacillus from Phylum firmicutes. Dairy wastewater (COD 27.73 g L-1, pH 11.3) at a temperature of 18±5°C was fed to a 1.5 L continuous fixed bed-reactor using Opuntia imbricata as substratum. Results obtained showed that at an HRT of 0.4 h the yield hydrogen production was 2.11 mmol H2 g-1 CODconsumed, mean while an increase in HRT to 0.8 h caused a decrease in yield hydrogen production to 1.31 mmol H2 g-1 CODconsumed.