A limited number of anti-tuberculosis drug candidates with novel mode of action have entered clinical trials in recent years. ATP synthase is one such validated drug target which has yielded a drug recently. The aim of this study was to identify the novel chemical scaffolds targeting the Mycobacterium tuberculosis (M. tuberculosis) ATP synthase. In this study, inverted membrane vesicles of Mycobacterium smegmatis were prepared to establish luciferin based ATP estimation assay. This assay was used to screen 700 compounds which were earlier found to be active on the whole cell of M. tuberculosis. Antibacterial activity of hits against various susceptible and drug-resistant strains of M. tuberculosis was evaluated using the microplate alamar blue assay and their cytotoxicity was also determined to select the safe compounds for further study. Screening of 700 compounds resulted in the identification of two compounds (5228485 and 5220632) exhibiting an IC₅₀ of 0.32 and 4.0 μg/ml respectively. Both compounds showed excellent anti-TB activity (MIC of 0.5–2.0 μg/ml against Mtb H₃₇Rv) and low cytotoxicity in human cell line and sub-mitochondrial particles. The three-dimensional structure of M. tuberculosis ATPase was predicted using in-silico approach and docking studies were performed with the active compounds. The interaction between compounds and bacterial ATP synthase was confirmed by molecular docking analysis. In conclusion screening of compound library has resulted in the identification of two novel chemical scaffolds targeting mycobacterial ATP synthase.