A series of gatifloxacin-1,2,3-triazole-isatin hybrids 8a-l were designed, synthesized, and screened for their in vitro antimycobacterial activity as well as cytotoxicity toward Chinese Hamster Ovary (CHO) cells. The synthesized hybrids showed considerable activity against MTB H Rv and two MDR-MTB strains with minimal inhibitory concentration (MIC) of 0.25-8 μg/ml, and the hybrid 8a (MIC : 0.25 μg/ml and MIC : 0.5 and 1 μg/ml) was found to be most active against the tested strains, which was not inferior to the parent gatifloxacin (MIC: 0.5, 0.25, and 0.5 μg/ml) against all three tested strains, and was ≥128-fold more active than isoniazid (MIC: ≥64 μg/ml) and rifampicin (MIC: >128 μg/ml) against the two MDR-MTB strains. Moreover, hybrid 8a (CC : 8.0 μg/ml) also displayed acceptable cytotoxicity toward CHO cells, and the selectivity index was 32. The structure-activity relationship and structure-cytotoxicity relationship were also enriched.