Twelve novel propylene-tethered ciprofloxacin-isatin hybrids 3a-f and 4a-f were designed, synthesized and characterized by MS, HRMS, 1H NMR and 13C NMR. All hybrids were evaluated for their in vitro antimicrobial activities against representative Gram-positive, Gram-negative and mycobacterial pathogens, cytotoxicity in VERO cell line as well as metabolic stability and in vivo pharmacokinetic (PK) properties. The preliminary results indicated that all mono-isatin-ciprofloxacin hybrids exhibited excellent antibacterial activities with MIC ranging from ≤0.03 to 0.5 μg/mL against most of the tested strains. In particular, ciprofloxacin-isatin hybrid 3d was highly potent against all tested Gram-positive and Gram-negative strains including clinically important drug-resistant pathogens, which was comparable to or more potent than the parent ciprofloxacin and reference levofloxacin. Whereas, conjugate 3b (MIC: 0.10 and 0.5 μg/mL) was 4- and 8-fold more active than ciprofloxacin (MIC: 0.78 μg/mL) and rifampicin (MIC: 0.39 μg/mL) against MTB H37Rv, and 4->256 times more potent than the three references ciprofloxacin (MIC: 2.0 μg/mL), rifampicin (MIC: 32 μg/mL) and isoniazid (>128 μg/mL) against MDR-TB. Both hybrid 3b and 3d with low cytotoxicity (CC50: 64 and 256 μg/mL) also showed acceptable metabolic stability and in vivo PK properties, could act as leads for further optimization.