Enterococcus mundtii, a commensal intestinal bacterium, was demonstrated to inhibit the growth of someMycobacterium tuberculosiscomplex (MTC) species that cause tuberculosis in humans and mammals. To further explore this preliminary observation, we cross-investigated fiveE. mundtiistrains and seven MTC strains representative of four MTC species using a standardized quantitative agar well diffusion assay. All fiveE. mundtiistrains, calibrated at 10 MacFarland, inhibited the growth of allM. tuberculosisstrains with various susceptibility profiles, but no inhibition was observed with lower inoculums. Further, eightE. mundtiifreeze-dried cell-free culture supernatants (CFCS) inhibited the growth ofM. tuberculosis,Mycobacterium africanum, Mycobacterium bovisandMycobacterium canettii, the most susceptible MTC species (inhibition diameter 25±1 mm), proportionally to CFCS protein concentrations. The data reported here indicate that theE. mundtiisecretome inhibited growth of all MTC species of medical interest, which broadens previously reported data. In the gut, theE. mundtiisecretome may modulate the expression of tuberculosis, exhibiting an anti-tuberculosis effect, with some protective roles in human and animal health.