Mycobacterial infections are set to surpass cancer as the leading cause of death by 2050. Treatment for antibiotic resistant infections is complicated and expensive. Many mycobacterial species are highly resistant to common antibiotics largely because of their unique cell wall, with a differentially crosslinked peptidoglycan (PG) layer. L,D-transpeptidase (Ldt) enzymes crosslink PG and are potential therapeutic targets for combatting mycobacterial infections. Six classes of Ldts have been identified, which are predicted to serve unique roles in PG biosynthesis. By better understanding the roles of Ldts, we can design novel antibiotics that target Ldts effectively and better treat deadly mycobacterial infections.