Mycobacterium habana was isolated in Cuba in 1971. Later, was demonstrated its protection capacity in mycobacterial infection. Here we determined the level of virulence, immunogenicity and the efficacy of three different M.habana strains as attenuated live vaccines. Intratracheal infection of BALB/c mice with high dose M.habana TMC 5135 or IPK-337 strains permitted 100% survival and limited tissue damage. Mice infected with M.habana IPK-220 showed lower attenuation, so it was discarded for the vaccination experiments. Strains IPK-337 and TMC 5135 were used as subcutaneous vaccine and compared with BCG. Nude mice vaccinated with strain 5135 showed longer but non-significant survival than BCG vaccinated animals. Cell suspensions from M.habana vaccinated mice produced higher IFNγ after stimulation with mycobacterial antigens than BCG recipients. After four months of challenge with Mycobacterium tuberculosis strain H37Rv, mice vaccinated with BCG substrain Phipps or strain TMC 5135 showed total survival, while 60% survival was exhibited by animals vaccinated with M.habana IPK-337. Both M.habana strains do not prevent the infection with M.tuberculosis but avoided the progression of the experimental disease; strain TMC 5135 showed similar level of protection than BCG.