Twelve novel 1H-1,2,3-triazole-tethered gatifloxacin (GTFX) isatin conjugates 5a-l with greater lipophilicity compared with GTFX were designed, synthesized and evaluated for their in vitro anti-mycobacterial activities against M. tuberculosis (MTB) H37Rv and MDR-TB as well as cytotoxicity. The preliminary results showed that all the targets (MIC: 0.10-8 μg/mL) exhibited excellent inhibitory activity against MTB H37Rv and MDR-TB, but eight of them (CC50: 7.8-62.5 μg/mL) were much more toxic than the parent GTFX (CC50: 125 μg/mL). Among them, 5g (MIC: 0.10 μg/mL) was 4-8 times more potent in vitro than the references GTFX (MIC: 0.78 μg/mL) and RIF (MIC: 0.39 μg/mL) against MTB H37Rv, but less active than INH (MIC: 0.05 μg/mL). The most potent 5g and 5h (MIC: 0.25 μg/mL) were 4->512 times more active than the three references (MIC: 1.0->128 μg/mL) against MDR-TB. Unfortunately, both of the two hybrids (CC50: 7.8 μg/mL) were much more cytotoxic than the other derivatives, need to be further optimized.