A series of isoniazid-amidoether derivatives was synthesized and screened for their antimycobacterial activity in vitro and in vivo. Most of the compounds exhibited potent in vitro activity against Mycobacterium tuberculosis H37Rv strain with MIC99 values ranging from 0.39 to 3.125 µM. Five compounds were equally potent to the reference compound isoniazid. The most active compound 3b, when evaluated for in vivo activity exhibited mild reduction in the bacillary load in lungs. However it showed better effect in spleen. All the compounds were also evaluated for their cytotoxicity against THP-1 cell line and no toxicity was observed up to 50 µM concentrations. The calculated ADMET parameters for the compounds validated good pharmacokinetic properties, confirming that these compounds could be used as a template for the development of new anti-tuberculosis agents.