In this paper, we report the synthesis and the structure–activity relationship study of three hydrazone analogs; the Schiff base hydrazone SBH and 2, 4-dinitrophenylhydrazones H1 & H2 derived from (E)-chalcones, to identify the active fragment of each structure. This identification has been carried out following in vitro biological evaluation, which revealed that the analogs H1 and H2 showed significant antibacterial activity due to their (E)-chalcone fragments characterized by proton NMR data and demonstrated by the docked view with emphasis on the involvement of these moieties in the interaction with the DNA gyrase, and thus contributes to the pharmacophore modeling. At the same time, SBH exhibited the highest free radical DPPH scavenging power associated with hydrogen bonding and conjugated push−pull chromophores, which were elucidated by reported vibrational assignments and absorption spectra. The DFT optimizations gave rise to non-planar and distorted structures around the hydrazone group with comparable geometrical parameters. The chemical descriptors predict comparable biological activities, while the BDE necessary for the H-abstraction indicated the best antioxidant activity for the Schiff base hydrazone SBH compound.