Abstract: Ciprofloxacin is one of fluoroquuinones widely used as antibiotics in clinical treatments. It has been shown that the carboxyl group on position 3 of ciprofloxacin is essential for antimicrobial activity, however, the amide form on this position and its corresponding biological effects have not been studied. To determine the structure-activity relationship, the ciprofloxacin hydrazide derivative was synthesized and its antimicrobial and antitumor mechanism was also evaluated preliminarily. Our results demonstrated that the substitution of -OH of carboxyl on position 3 of ciprofloxacin with a -NH-NH₂ group could slightly alter the antimicrobial spectra, but not significantly. The studies of the hydrazide derivative on cell cycle arrest, mitochondrial membrane permeability, topoisomerase inhibition, pro-apoptotic gene regulation and molecular docking revealed that the minor structural modification on position 3 of ciprofloxacin did not result in changes in molecular targets compared to ciprofloxacin.