Biohydrogen production by dark fermentation (DF) can be achieved using only indigenous bacteria from substrates or by adding an external inoculum. This study aims to provide new insights on the role of indigenous bacteria in DF process operation by investigating DF performances and microbiological aspects. DF tests are performed with only indigenous bacteria, with indigenous and exogenous bacteria and with only exogenous bacteria by inactivating indigenous bacteria by gamma irradiation. Sorghum irradiation reduces DF performances for both hydrogen (17.8 ± 12.8 versus 45.2 ± 1.7 mLH 2 /gVS added ) and total metabolite (0.22 ± 0.01 versus 0.30 ± 0.01 gCOD/gVS added ) yields. In contrast, no difference is observed with the organic fraction of municipal solid waste, suggesting a distinct role of indigenous bacteria for both substrates. Indigenous bacteria inactivation strongly modifies the metabolic routes and final bacterial community composition. This study proves the key role of indigenous bacteria in influencing metabolite production and bacterial composition.