A series of novel ciprofloxacin (CP) derivatives substituted at the N-4 position with biologically active moieties were designed and synthesised. 14 compounds were 1.02- to 8.66-fold more potent than doxorubicin against T-24 cancer cells. Ten compounds were 1.2- to 7.1-fold more potent than doxorubicin against PC-3 cancer cells. The most potent compounds 6, 7a, 7b, 8a, 9a, and 10c showed significant Topo II inhibitory activity (83–90% at 100 μM concentration). Compounds 6, 8a, and 10c were 1.01- to 2.32-fold more potent than doxorubicin. Compounds 6 and 8a induced apoptosis in T-24 (16.8- and 20.1-fold, respectively compared to control). This evidence was supported by an increase in the level of apoptotic caspase-3 (5.23- and 7.6-fold, sequentially). Both compounds arrested the cell cycle in the S phase in T-24 cancer cells while in PC-3 cancer cells the two compounds arrested the cell cycle in the G1 phase. Molecular docking simulations of compounds 6 and 8a into the Topo II active site rationalised their remarkable Topo II inhibitory activity.