A series of novel gemifloxacin (GMFX) derivatives containing a substituted benzyloxime moiety with remarkable improvement in lipophilicity were synthesized. The target compounds evaluated for their in vitro antibacterial activity against representative strains. Our results reveal that most of the target compounds have considerable potency against all of the tested gram-positive strains including MRSA and MRSE (MIC: <0.008-8 μg/mL), although they are generally less active than the references against the gram-negative strains. In particular, compound 11l (MIC: <0.008-4 μg/mL) was found to be 8-2048 and 2-128 times more potent than levofloxacin (LVFX) and GMFX against the gram-positive strains, respectively. Moreover, against MRSA clinical isolates, 11l (MIC(90): 1 μg/mL) is 8-fold more active than GMFX, and 2-fold more active than GMFX and moxifloxacin against MRSE clinical isolates (MIC(90): 4 μg/mL).