The World Health Organization has put drug resistance in tuberculosis on its list of significant threats, with a critical emphasis on resolving the genetic differences in Mycobacterium tuberculosis. This provides an opportunity for a better understanding of the evolutionary progression leading to anti-microbial resistance. Anti-microbial resistance has a great impact on the economic stability of the global healthcare sector. We performed a timeline genomic analysis from 2003 to 2021 of 578 mycobacterium genomes to understand the pattern underlying genomic variations. Potential drug targets based on functional annotation was subjected to pharmacophore-based screening of FDA-approved phyto-actives. Reaction search, MD simulations, and metadynamics studies were performed. A total of 4,76,063 mutations with a transition/transversion ratio of 0.448 was observed. The top 10 proteins with the least number of mutations were high-confidence drug targets. Aminoglycoside 2’-N-acetyltransferase protein (AAC2’), conferring resistance to aminoglycosides, was shortlisted as a potential drug target based on its function and role in bait drug synergism. Gentamicin-AAC2’ binding pose was used as a pharmacophore template to screen 10,570 phyto-actives. A total of 66 potential hits were docked to obtain naloxone as a lead — active with a docking score of -6.317. Naloxone is an FDA-approved drug that rapidly reverses opioid overdose. This is a classic case of a repurposed phyto-active. Naloxone consists of an amine group, but the addition of the acetyl group is unfavorable, with a reaction energy of 612.248 kcal/mol. With gentamicin as a positive control, molecular dynamic simulation studies were performed for 200 ns to check the stability of binding. Metadynamics-based studies were carried out to compare unbinding energy with gentamicin. The unbinding energies were found to be −68 and −74 kcal/mol for naloxone and gentamycin, respectively. This study identifies naloxone as a potential drug candidate for a bait drug synergistic approach against Mycobacterium tuberculosis.