The aim of this study was to determine how bio-hydrogen production was related to the composition of the bacterial community in a dark fermentation fed with marine brown algae (Laminaria japonica). The bacterial diversity was ascertained by 16S rDNA PCR-sequencing. A total of 444 mL of bio-hydrogen was produced from 10 g/L of dry algae in a 100 mL of culture fluid for 62 h. The pH varied from 8.74 to 7.05. Active bio-hydrogen production was observed from 24 to 48 h, and maximum bio-hydrogen production was 106 mL over 1 L gas. The bacterial community of the activated sludge consisted of 6 phyla, where H2 producing and consuming bacteria coexisted. The only detectable bacterial phylum after bio-hydrogen generation with heat-treated (65 ℃, 20 min) seeding was Firmicutes. Clostridium and Bacillus species constituted 54% and 46%, respectively, of the bacterial mixture and the most abundant species was Clostridium beijierinckii (34%). These results may provide a better understanding of how different biohydrogen communities affect hydrogen production and aid in the optimization of bio-hydrogen production.