A novel derivative of ciprofloxacin (Cpx) was synthesized and characterized using various analytical techniques, including FT-IR spectroscopy, UV-Vis spectroscopy, TEM and SEM analysis, 1H NMR, 13C NMR, and HPLC analysis. The newly prepared Cpx derivative (Cpx-Drv) exhibited significantly enhanced antibacterial properties compared to Cpx itself. In particular, Cpx-Drv demonstrated a 51% increase in antibacterial activity against S. aureus and a 30% improvement against B. subtilis. It displayed potent inhibitory effects on topoisomerases II (DNA gyrase and topoisomerase IV) as potential molecular targets, with IC50 values of 6.754 and 1.913 µg/mL, respectively, in contrast to Cpx, which had IC50 values of 2.125 and 0.821 µg/mL, respectively. Docking studies further supported these findings, showing that Cpx-Drv exhibited stronger binding interactions with the gyrase enzyme (PDB ID: 2XCT) compared to the parent Cpx, with binding affinities of −10.3349 and −7.7506 kcal/mole, respectively.