Animal mycobacterioses are among the most important zoonoses worldwide. These are generally caused by either Mycobacterium tuberculosis (MTB), M. bovis (MBO) or M. avium (MAV). To test the hypothesis that different species of pathogenic mycobacteria isolated from varied anatomic locations or animal species differ in virulence and pathogenicity, we performed experiments with three mycobacteria strains (NTSE-3(MTB), NTSE-4(MBO) and NTSE-5 (MAV)) obtained from animal species. Spoligotyping analysis was used to confirm both MTB and MBO strains while the MAV strain was confirmed by 16s rDNA sequencing. BALB/c mice were intranasally infected with the three strains at low and high CFU doses to evaluate variations in pathogenicity. Clinical and pathological parameters were assessed. Infected mice were euthanized at 80 days post-inoculation (dpi). Measures of lung and body weights indicated that the MBO infected group had higher mortality, more weight loss, higher bacterial burden and more severe lesions in lungs than the other two groups. Cytokine profiles showed higher levels of TNF-α for MBO versus MTB, while MAV had the highest amounts of IFN-β in vitro and in vivo. In vitro levels of other cytokines such as IL-1β, IL-10, IL-12, IL-17, and IFN-β showed that Th1 cells had the strongest response in MBO infected mice and that Th2 cells were inhibited. We found that the level of virulence among the three isolates decreased in the following order MBO>MTB>MAV.