The emerging resistance to currently available antimicrobial drug demands the synthesis of new agents for microbial infections. In the current research work, a novel series of N'-[2-(substituted sulfanyl)quinazolin-4-yl] pyridine-4 carbohydrazide derivatives (5D1-5D4) were synthesized and characterized by IR, 1H NMR, Mass spectra and elemental analysis. All the newly prepared molecules were tested for their antibacterial and antifungal activity against six strains of gram positive and gram-negative bacteria and two strains of fungi using serial plate dilution method. Among the prepared products, N'-{2-[(2-fluorophenyl) sulfanyl] quinazolin-4-yl} pyridine-4-carbohydrazide 5D1 was found to exhibit the highest in vitro antibacterial and antifungal potential with minimum inhibitory concentration (MIC) values of 3.12, 0.4, 0.8, 12.5, 12.5, 6.25, 0.8 and 1.6 μg/ml against the respective strains. The MIC of compounds 5D2 and 5D4 were found comparable with fluconazole, making it most potent antimicrobial agents in the series. In the present investigation all the compounds were found to exhibit more inhibition towards gram positive than gram negative bacterial strains. Molecular docking was performed to dock compounds into the ecKAS III binding site, which suggested probable inhibition mechanism. The results revealed the higher potency of compounds 5D1, 5D3 and 5D4 based on glide gscore and binding poses of the molecules as ecKAS III synthase inhibitors.