Owing to the rise in drug resistance in tuberculosis combined with the global spread of its causative pathogen, Mycobacterium tuberculosis (Mtb), innovative anti-mycobacterial agents are urgently needed. To address this problem, we have employed a drug repurposing approach to discover novel FDA-approved drugs to inhibit Mtb growth. Here, we have used essential Mtb enzyme, DNA gyrase, a promising and potential target for novel anti-tuberculosis chemotherapeutics. High-throughput screening of compounds (using FDA-compounds library) was done against the active site of Mtb DNA gyrase, the region of ATP binding (N-terminal domain) pocket on gyrase B subunit. Here, we identified a total of four compounds (Drug97, Drug45, Drug77, and Drug38) tightly binds to ATPase binding pocket of N-terminal domain of gyrase B (MtbGyrB47). We investigated the binding activity of identified drugs using various biophysical techniques such as thermal denaturation (CD Spectroscopy), Fluorescence titration (Fluorescence Spectroscopy) and Surface Plasmon Spectroscopy (SPR) and calculated IC50 values of these drugs using EMSA, Supercoiling, and ATPase assay. Among which, Drug97, an anthracycline antibiotic (used as an anticancer drug), was found to be a potent inhibitor of Mtb DNA gyrase. Low-μM inhibition of Mtb DNA gyrase was correlated with their low-μM minimum inhibitory concentrations for all screened FDA-drugs. Drug97 exhibited IC50 value of 0.6±0.14 µM against MtbGyrB47, kD values of 0.06±0.21 µM and MIC90 values of 0.12 µg/ml. Our results strongly suggest that the screened compounds (anthracyclines) target mycobacterial DNA gyrase, inhibits gyrase catalytic cycle.