A series of novel 7-(3-alkoxyimino-4-amino-4-methylpiperidin-1-yl)fluoroquinolone derivatives were designed, synthesized and evaluated for their in vitro antibacterial activity and cytotoxicity. All of the target compounds have potent antibacterial activity against the tested Gram-positive and Gram-negative strains, and exhibit good potency in inhibiting the growth of Staphylococcus aureus including MRSA, Staphylococcus epidermidis including MRSE and Streptococcus pneumoniae (MICs: 0.125-4 μg/mL). Compound 22, with the best activity against Gram-positive strains, is 4-16 fold more potent than gemifloxacin, gatifloxacin and levofloxacin against Enterococcus faecalis, and 16- and 4-fold more potent than levofloxacin against S. epidermidis 09-6 and S. pneumoniae 08-4, respectively.