Pazufloxacin is a broad-spectrum synthetic fluoroquinolone antibiotic. It is active against Gram-negative and Gram-positive bacteria, including clinical isolates of E. coli, K. pneumoniae, methicillin-susceptible and -resistant S. aureus, and methicillin-susceptible and -resistant S. epidermidis (MIC90s = 0.05, 0.1, 0.39, 12.5, 0.39, and 6.25 μg/ml, respectively). It inhibits E. coli, P. aeruginosa, and S. aureus DNA gyrase (IC50s = 0.88, 1.9, and 10.2 μg/ml, respectively) and S. aureus topoisomerase IV (IC50 = 24.2 μg/ml) in cell-free assays. In vivo, pazufloxacin is active against systemic E. coli, K. pneumoniae, and methicillin-resistant S. aureus infections in mice (ED50s = 0.15, 5.5, and 4.5 mg/kg, respectively).