DNA gyrase of Mycobacterium tuberculosis (MTB) is a type II topoisomerase and is a well-established and validated target for the development of novel therapeutics. By adapting the medium throughput screening approach, we present the discovery and optimization of ethyl 5-(piperazin-1-yl) benzofuran-2-carboxylate series of mycobacterial DNA gyraseB inhibitors, selected from Birla Institute of Technology and Science (BITS) database chemical library of about 3000 molecules. These compounds were tested for their biological activity; the compound 22 emerged as the most active potent lead with an IC50 of 3.2±0.15μM against Mycobacterium smegmatis DNA gyraseB enzyme and 0.81±0.24μM in MTB supercoiling activity. Subsequently, the binding of the most active compound to the DNA gyraseB enzyme and its thermal stability was further characterized using differential scanning fluorimetry method.