Hydrogen production by biological methods, particularly dark fermentation, has recently gained in interest for the scientific as well as the socio-economic communities. This theme has been intensively investigated over the past decade since biohydrogen can be produced under a large range of conditions. This chapter aims to present the basic concepts of biohydrogen production by dark fermentation, including microbial metabolism and related microorganisms. The wide range of substrate possibilities, as well as the most important operational parameters, are also reviewed. Integration of the dark fermentation bioprocesses into the concept of environmental biorefinery is further discussed by proposing alternatives to overcome the limitations prior to their application at industrial scale. Finally, the current situation of hydrogen as main energetic vector for the future, as well as the role of dark fermentation in the environmentally friendly hydrogen production are discussed.