A series of novel balofloxacin ethylene isatin derivatives with remarkable improvement in lipophilicity, as compared to the parent compound balofloxacin, were designed, synthesized and characterized by (1)H NMR, MS and HRMS. These derivatives were initially evaluated for their in vitro antimycobacterial activity against M. phlei CMCC 93201 and M. smegmatis CMCC 93202. Compounds 3b, 3d, 3g-j and 3l were chosen for further evaluation their in vitro activity against MTB 09710 clinical isolate, and then compounds 3h and 3g against MTB H37Rv ATCC 27294. All of the synthesized compounds were less active than balofloxacin against M. phlei CMCC 93201 and M. smegmatis CMCC 93202, but compounds 3g-j (MIC: <0.5-8 microg/mL) were more potent than balofloxacin (MIC: 16 microg/mL) against MTB 09710. In particular, compound 3h (MIC: 0.25- < 0.5 microg/mL) was found to be comparable to moxifloxacin, and >or=32 fold more potent than balofloxacin against MTB 09710 and MTB H37Rv ATCC 27294. The results demonstrated that the lipophilicity of the tested compounds was not the sole parameter affecting antimycobacterial activity, as well as the potential and importance of developing new fluoroquinolone derivatives against mycobacterial infections.