Tuberculosis (TB) an infectious disease with very high mortality claims approximately 1.5 million lives and infects 2 billion people annually. The present study evaluated anti-tuberculosis activity of benzo(c)thiophene-1,3-dione against Mycobacterial tuberculosis H37RV in vitro and in vivo in tuberculosis mice model. The MIC of benzo(c)thiophene-1,3-dione, rifampicin and isoniazid were found to be 4.0, 2.0 and 1.0 μg/ml, respectively. Benzo(c)thiophene-1,3-dione showed MIC in the range of 8-14 μg/ml against four drug-resistant isolates of M. tuberculosis and MBC 14 μg/ml against M. tuberculosis H37RV. Interaction of benzo(c)thiophene-1,3-dione with rifampicin led to 2-fold increase in anti-TB activity whereas with isoniazid improvement showed 4-fold enhancement. Fractional inhibitor concentration index suggested additive interaction with rifampicin and synergism with isoniazid. Treatment with benzo(c)thiophene-1,3-dione at 1X MIC indicated bacteriostatic activity whereas at 2X, 4X and 4X MIC doses significant reduction in M. tuberculosis load was observed. The benzo(c)thiophene-1,3-dione administration to mice at doses of 5000 and 1000 mg/kg caused no changes in behaviour nor any death. Benzo(c)thiophene-1,3-dione treatment of tuberculosis mice model effectively inhibited pulmonary CFU compared to model group. Data obtained from MTT assay showed negligible cytotoxicity of benzo(c)thiophene-1,3-dione against CMMT, MB 157, CL-S1, normal breast cells in 5-320 μg/ml concentration range. Thus, benzo(c)thiophene-1,3-dione exhibits promising anti-mycobacterial activity against Mycobacterium tuberculosis H37RV and other drug resistant strains. In Mycobacterium tuberculosis mice model benzo(c)thiophene-1,3-dione significantly suppressed bacterial load and showed synergism with isoniazid. Therefore, benzo(c)thiophene-1,3-dione has potential to be evaluated further for development of anti-tuberculosis treatment.Copyright © 2020. Published by Elsevier Ltd.