A number of novel 1, 3, 4-oxadiazole analogues have been designed and synthesized by Condensation of substituted aldehyde/ketone with substituted benzohydrazide to form substituted N'-alkylidene benzohydrazide and then cyclization of N'-alkylidene benzohydrazide to form 1, 3, 4-oxadiazole derivatives. To investigate the antimicrobial data on structural basis, in-silico docking studies of the synthesized compounds (4a-4r) into the crystal structure of E-coli DNA gyrase (Type-2 topoisomerase) using Autodock PyRx virtual screening program were performed to predict the affinity and orientation of the synthesized compounds at the activities by using 6rks PDB. Inhibiting the ATPase activity of gyrase blocks the introduction of negative supercoils in DNA and traps the chromosome in a positively supercoiled state that may have a downstream impact on cell physiology and division. The results indicate that ketone substituted benzohydrazide derivatives show good binding affinity (-8kcal to -9kcal) and electron-withdrawing group such as –NO₂ and -Cl present at R1 increases the affinity of scaffold and DNA gyrase receptors and binds into the specificity pocket. In this pocket, the 1, 3, 4-oxadiazole nucleus of these compounds interacts with the amino acid Alanine A: 421, Valine A: 420, Tyrosine A: 478 and Glutamine A: 381 residues of the target. Also, it is verified by in vitro antimicrobial screening, where all the compounds were active against tested bacterial strains. Among these compounds 4(c), 4(d), (4e), (4h), (4i), 4(m), 4(n), 4(o), 4(p), and (4q) showed good bacterial zone inhibition.