Herein we report the design and synthesis of a series of novel benzofuran-isatin hybrids, and in vitro evaluation of their anti-mycobacterial activity against both drug-susceptible and multi-drug resistant (MDR) Mycobacterium tuberculosis (MTB) strains. In parallel, cytotoxicity of these hybrids was also tested in VERO cells. Preliminary results indicated that all hybrids with acceptable cytotoxicity in VERO cells (CC50: 128->1024 μg/mL) exhibited considerable anti-mycobacterial activities against MTB H37Rv and MDR-TB with MIC ranging from 0.25 to 8 μg/mL. It is worth noting that hybrid 8f with no cytotoxicity towards VERO cells (CC50: >1024 μg/mL) was found to be the most active compound (MIC: 0.25 and 0.5 μg/mL) against MTB H37Rv and MDR-TB strains. Comparing to the first-line anti-tuberculosis agents rifampicin and isoniazid, hybrid 8f has shown over two magnitude more active against MDR-TB. The hybrid 8f was further evaluated for its metabolic stability and in vivo pharmacokinetic profiles, with the results showing that hybrid 8f exhibited lower metabolic stability compared to inhibitor TAM16. Further modification based on hybrid 8f is needed to improve the metabolic stability and pharmacokinetic profiles.