The fermentative production of H-2 from residual glycerol is an attractive alternative for clean energy production from a waste product. Selection of operational variables for microbial populations with an adequate diversity in order to improve H-2 yields is an issue faced during optimization of biological production of H-2. Operational and environmental factors affect both microbial diversity and the activity of specific enzymes. Therefore, these variables must be controlled to obtain the best H-2 yields. This review covers the main variables involved in the fermentative production of H-2 from crude glycerol and the biochemistry of the anaerobic digestion of glycerol, with a focus on the microbial diversity involved in this process.