Few quinoline-based synthetic compounds (2, 8 Dicyclopentyl-4-methyl quinoline and 2, 8 Dicyclohexyl-4-methyl quinoline), the synthesis of which have been already shown by our medicinal chemistry group, were found to be potent inhibitor of mycobacterial growth. Based on the results of cell culture-based cell killing assays using DNA gyrase positive E. coli strains, we presumed that bacterial DNA gyrase might be a probable target of quinolines. The resemblance of the basic skeletal structural moiety of quinolone and quinoline inspired us to hypothesize that these quinolines might inhibit DNA gyrase. While the non-gyrase inhibitors like ethambutol and isoniazid did not inhibit the growth of these strains. The genesis of the notion of using E. coli DNA gyrase as an alternative to DNA gyrase from the pathogenic Mycobacterium, stems from the fact that E. coli DNA gyrase is found to be about eighty times more sensitive to the action of quinolones than the Mycobacterium DNA gyrase. Therefore, we had used E. coli DNA gyrase as a model enzyme for studying the action of some synthetic quinoline compounds synthesized by us. In the present work, we have used cell killing assay, gel electrophoresis assay (for DNA supercoiling) and UV spectroscopy-based coupled assay (for ATP hydrolysis) for characterizing the activity of DNA gyrase. Quinolones exhibited low IC50 values as compared to the studied quinolines on DNA gyrase positive E. coli strains We found that although quinolones are the potent inhibitors of supercoiling activity of E. coli DNA gyrase, quinolines are not. We further found that ATPase activity of E. coli DNA gyrase (Non-specific inhibitor) was inhibited to a very minor extent in the presence of very high concentration of these synthetic quinolines. DNA gyrase is not the primary target of these synthetic quinolines (2, 8 Dicyclopentyl-4-methyl quinoline and 2, 8 Dicyclohexyl-4-methyl quinoline).