A series of novel fluoroquinolone derivatives containing an 3-alkoxyimino-4-(cyclopropylanimo)methylpyrrolidine moiety were designed, synthesized and evaluated for their biological activity. Our results revealed that 19b2 shows good activity against MTB H37Rv ATCC 27294 (MIC: <0.25 μg/mL) and MDR-MTB 6133 clinical isolate (MIC: 0.11 μg/mL). Most of them have potent potency against Gram-positive strains, although they are generally poor active against Gram-negative strains. Especially, compounds 22b1 and 23a3 (MICs: <0.008-8 μg/mL) were found to 2-128 times more potent than ciprofloxacin and levofloxacin against all of the tested Gram-positive strains including quinolone-resistant MRSA, MRSE, Enterococcus faecium and Enterococcus faecalis.