This paper mainly analyzes the effect of oxidizing agents on biohydrogen production from Clostridium acetobutylicum; however, since various oxidoreduction reactions are involved in the production of biohydrogen in dark fermentation, only the effect of dichloro dicyano benzo quinine (DDQ) as an oxidizing agent was investigated here. Results show that the addition of 0.02 g/L DDQ leads to an increase of hydrogen production rate and yield by 25and 19%, respectively. It was also observed that the amount of undesired end products like ethanol, butanol, and propionate was reduced in the presence of DDQ and the acetate to butyrate ratio (A/B) increased from 0.82 to 1.31 on the addition of DDQ.