Toxin-antitoxin (TA) systems are highly conserved in members of the Mycobacterium tuberculosis (Mtb) complex and have been proposed to play an important role in physiology and virulence. Nine of these TA systems belong to the mazEF family, encoding the intracellular MazF toxin and its antitoxin, MazE. By overexpressing each of the nine putative MazF homologues in Mycobacterium bovis BCG, here we show that Rv1102c (MazF3), Rv1991c (MazF6) and Rv2801c (MazF9) induce bacteriostasis. The construction of various single-, double- and triple-mutant Mtb strains reveals that these MazF ribonucleases contribute synergistically to the ability of Mtb to adapt to conditions such as oxidative stress, nutrient depletion and drug exposure. Moreover, guinea pigs infected with the triple-mutant strain exhibits significantly reduced bacterial loads and pathological damage in infected tissues in comparison with parental strain-infected guinea pigs. The present study highlights the importance of MazF ribonucleases in Mtb stress adaptation, drug tolerance and virulence.