A novel series of Schiff bases 5b-g was synthesized by the reaction of N-substitutedbenzylisatin 3b-g with nalidixic acid hydrazide 4 in order to optimize the antimycobacterial activity of our lead Schiff base 5a. Antimycobacterial (anti-mtb) activity of the synthesized hydrazones was investigated against four Mycobacterium strains: M. intercellulari, M. xenopi, M. cheleneo and M. smegmatis. It was found that para-substitution, with electron withdrawing group, of benzyl moiety in N-benzylisatins resulted in 7 fold enhancement of the anti-mtb activity as shown with compounds 5b, 5d and 5f (MIC 0.09 μg/ml) with p-chloro, fluoro and nitro substituents respectively. In silico docking study of these hydrazones with mtb-DNA gyrase revealed that there is parallelism between the antimycobacterial activity of these hydrazones and docking with the active site of the mtb-DNA gyrase B subunit.