A series of benzofuran-isatin hybrids 6a-n and 7a-g linked by alkyl linkers were designed and synthesized. Among them, hybrids 6a-l and 7a-g were assessed for their in vitro anti-mycobacterial activities against two multi-drug resistant Mycobacterium tuberculosis (MDR-MTB) strains and the cytotoxicity towards CHO cells. The preliminary results indicated that all hybrids (MIC: 0.125-16 μg/mL) showed excellent activity against the tested MDR-MTB strains, and low cytotoxicity (CC<sub>50</sub>: 64->512 μg/mL) towards CHO cells. Among them, hybrid 7e (MIC: 0.125 and 0.25 μg/mL) was highly active against the tested two MDR-MTB strains, which was 8-16 folds better than ciprofloxacin (MIC: 1 and 4 μg/mL), ≥512 folds more potent than rifampicin (MIC: 64 and > 128 μg/mL) and isoniazid (MIC: >128 μg/mL), but it was less active than TAM16 (MIC: <0.06 μg/mL). Moreover, the hybrid 7e (CC<sub>50</sub>: 128 μg/mL) also showed low cytotoxicity towards CHO cells, and high selectivity index (1,024). However, the metabolic stability and in vivo pharmacokinetic profiles of hybrid 7e were inferior to TAM16, so it still needs to be modified so as to get the optimized hybrid for potential use in mycobacterial treatment.