A novel series of N-acylated ciprofloxacin (CP) conjugates 1–21 were synthesized and screened as potential antimicrobial agents. Conjugates 1 and 2 were 1.25–10-fold more potent than CP toward all Staphylococci (minimal inhibitory concentration 0.05–0.4 μg/mL). Most of the chloro- (3–7), bromo- (8–11), and CF3-alkanoyl (14–16) derivatives expressed higher or comparable activity to CP against selected Gram-positive strains. A few CP analogues (5, 10, and 11) were also more effective toward the chosen clinical Gram-negative rods. Conjugates 5, 10, and 11 considerably influenced the phases of the bacterial growth cycle over 18 h. Additionally, compounds 2, 4–7, 9–12, and 21 exerted stronger tuberculostatic action against three Mycobacterium tuberculosis isolates than the first-line antitubercular drugs. Amides 1, 2, 5, 6, 10, and 11 targeted gyrase and topoisomerase IV at 2.7–10.0 μg/mL, which suggests a mechanism of antibacterial action related to CP. These findings were confirmed by molecular docking studies. In addition, compounds 3 and 15 showed high antiproliferative activities against prostate PC3 cells (IC50 2.02–4.8 μM), up to 6.5–2.75 stronger than cisplatin. They almost completely reduced the growth and proliferation rates in these cells, without a cytotoxic action against normal HaCaT cell lines. Furthermore, derivatives 3 and 21 induced apoptosis/necrosis in PC3 cells, probably by increasing the intracellular ROS amount, as well as they diminished the IL-6 level in tumor cells.
