This paper reports the susceptibility profile to rifabutin (RFB) 1 and six recently synthesized RFB analogs 3-8, of either rifampicin (RFP) susceptible Mycobacterium tuberculosis and resistant clinical isolates from two sources: Mexico and Brazil. Taking into account that about 95% of M. tuberculosis strains resistant to RFP present mutations in the rpoB gene, with some of these mutations being determinant also to RFB resistance, the RFB analogs were screened for activity against a set of known RFP susceptible and resistant strains. N'-Acetyl-RFB 5 and N'-(undec-10″-enoyl)-RFB 8 showed the best results, in particular with mutations in the codon 516, 522 and 531 of the rpoB gene, and were therefore selected for in vivo assessment of their efficacy. Studies conducted with tuberculous Balb/C mice previously infected with Ser531Leu mutated clinical isolate, evidenced both 5 and 8 as promoters of a significant decrease on tubercle bacilli burden in lungs associated with lower tissue damage, thus confirming them as good leads for drug discovery. The SAR of the acylated compounds 5 and 8 envisaging the identification of pharmacophore features, highlights the importance of profiling more clearly the chemistry within the molecular aspects for elucidation of the mode of action of RFB and analogs, in relation to mutations in Multidrug-Resistant (MDR) strains.