A series of novel 7-(3-alkoxyimino-4-methyl-4-methylaminopiperidin-1-yl)fluoroquinolone derivatives were designed, synthesized, and characterized by 1H-NMR, MS, and HRMS. These fluoroquinolones were evaluated for their in-vitro antibacterial activity against representative Gram-positive and Gram-negative strains. Generally, all of the target compounds have considerable antibacterial activity against the tested forty strains, and exhibit exceptional potency in inhibiting the growth of methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) ATCC33591 (MICs: 0.06 to 2 microg/mL). In particular, compounds 14, 19, 28, and 29 are fourfold more potent than ciprofloxacin against MSSA 08-49. Compounds 23, 26, and 27 are twofold more potent than ciprofloxacin against MRSA ATCC33591 and MSSA ATCC29213. In addition, compound 14 exhibits excellent activity (MIC: 0.06 microg/mL) against Acinetobactes calcoaceticus, which is two- to 16-fold more potent than the reference drugs gemifloxacin, levofloxacin, and ciprofloxacin.