We report herein the synthesis and antimycobacterial activity of 1,2,3-triazole derivatives of isoniazid. Most of the compounds exhibited potent activity against Mycobacterium tuberculosis H37Rv strain with MIC99 values ranging from 0.195 to 1.56 μM in vitro. One compound showed better in vitro activity than the reference, whereas five compounds were equally potent to the reference compound isoniazid. The cytotoxicity of these compounds was studied against THP-1 cell line and no toxicity was observed even at 50 μM concentration. The compound with most potent in vitro activity was evaluated for in vivo in murine model of tuberculosis and significantly reduced bacillary load in both lungs and spleen at 10 weeks post-treatment. However this clearance effect was more pronounced in the case of spleen. Molecular docking and molecular dynamics simulations have been performed using two targets 2IDZ 1 (wild type Enoyl-acyl-carrier-protein reductase) and 4DQU 2 (mutant type Enoyl-acyl-carrier-protein reductase) to study the binding orientation and stability of the compound 47. Docking studies proved compound 47 fit well into the binding pocket of both the targets. Molecular dynamic simulations concluded that the highest active compound 47 in complex with 4DQU was more stable when compared to the 2IDZ. We believe that further optimization of these molecules may lead to potent anti-tubercular agents.