Hydrogen is the lightest element and is an excellent energy carrier due to high energy intensity. Hydrogen can be produced in a number of ways like physical, chemical and biological methods. Since the physical and chemical method of hydrogen production is energy intensive, research is currently concentrated on the biological methods. Among the biological method dark fermentation has gained greater attention because of its independence to light and the ability to use various substrates for fermentation. In this review, there is detailed information about biohydrogen production by dark fermentation using microorganisms including, the hydrogen producing anaerobic and facultative anaerobic bacteria, the enzymes involved, the factors affecting the process such as temperature, pH, substrates, hydraulic retention time and hydrogen partial pressure. Also a brief discussion about the metabolic engineering aspects in biohydrogen production and a note on microbial electrolysis cell has been discussed.