The fermentative production of H2 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 H2 yields is an issue faced during optimization of biological production of H2. Operational and environmental factors affect both microbial diversity and the activity of specific enzymes. Therefore, these variables must be controlled to obtain the best H2 yields. This review covers the main variables involved in the fermentative production of H2 from crude glycerol and the biochemistry of the anaerobic digestion of glycerol, with a focus on the microbial diversity involved in this process.