Glycerol and glycerol-containing compounds such as lipids belong to the most abundant organic compounds that may serve as nutrient for many bacteria. For the cell wall-less bacteria of the genus Mycoplasma, glycerol derived from phospholipids of their human or animal hosts is the major source of carbon and energy. The lipids are first degraded by lipases, and the resulting glycerophosphodiesters are transported into the cell and cleaved to release glycerol-3-phosphate. Alternatively, free glycerol can be transported, and then become phosphorylated. The oxidation of glycerol-3-phosphate in Mycoplasma spp. as well as in related firmicutes involves a hydrogen peroxide-generating glycerol-3-phosphate oxidase. This enzyme is a key player in the virulence of Mycoplasma spp. as the produced hydrogen peroxide is one of the major virulence factors of these bacteria. In this review, the different components involved in the utilization of lipids and glycerol in Mycoplasma pneumoniae and related bacteria are discussed.