The E/Z isomers of butyl-2-phenyl-2-(2-phenylhydrazono)acetate were synthesized by us, and their structures were determined using ¹H and ¹³C NMR spectroscopy. Their biological activities were investigated both in vitro and through in silico methods. NMR analysis revealed that the Z-isomer exhibits strong intramolecular hydrogen bonding, which enhances its conformation and stability. In in vitro antibacterial tests, the Z-isomer showed stronger activity compared to the E-isomer. However, in silico docking results did not reveal significant differences between the isomers. Bioinformatic analyses demonstrated the potential activity of the molecules against various pathogenic bacteria, including Enterococcus faecium, Mycobacterium tuberculosis, and Streptococcus viridans. These results indicate that E/Z isomerism plays a crucial role in biological activity, and it is important to rely not only on virtual models but also on real biological experiments.