Bacteria belonging to the genus Mycobacterium can cause several infectious diseases affecting humans and animals. Here, we reviewed the latest advances in the development of DNA vaccines against TB, Buruli ulcer and Johne's disease. Current understanding of the immunity to the respective causative pathogens indicates that the use of DNA vaccines encoding mycobacterial antigens could lead to efficient vaccination strategies. Moreover, characterization of protective mycobacterial antigens has been greatly facilitated by the analysis of immune responses induced after DNA vaccination. In addition, work aiming at optimizing DNA vaccines against mycobacterial diseases and research related to the controversial development of postexposure and therapeutic DNA vaccines are also discussed.