Recent work has shed light on the abundance and diversity of D-amino acids in bacterial extracellular/periplasmic molecules, bacterial cell culture, and bacteria-rich environments. Within the extracellular/periplasmic space, D-amino acids are necessary components of peptidoglycan, and disruption of their synthesis leads to cell death. As such, enzymes responsible for D-amino acid synthesis are promising targets for antibacterial compounds. Further, bacteria are shown to incorporate a diverse collection of D-amino acids into their peptidoglycan, and differences in D-amino acid incorporation may occur in response to differences in growth conditions. Certain D-amino acids can accumulate to millimolar levels in cell culture, and their synthesis is proposed to foretell movement from exponential growth phase into stationary phase. While enzymes responsible for synthesis of D-amino acids necessary for peptidoglycan (D-alanine and D-glutamate) have been characterized from a number of different bacteria, the D-amino acid synthesis enzymes characterized to date cannot account for the diversity of D-amino acids identified in bacteria or bacteria-rich environments. Free D-amino acids are synthesized by racemization or epimerization at the α-carbon of the corresponding L-amino acid by amino acid racemase or amino acid epimerase enzymes. Additionally, D-amino acids can be synthesized by stereospecific amination of α-ketoacids. Below, we review the roles of D-amino acids in bacterial physiology and biotechnology, and we describe the known mechanisms by which they are synthesized by bacteria.