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Zabofloxacin is a novel fluoroquinolone agent that has potent activity against gram-positive pathogens. In this study, we confirmed that zabofloxacin showed the most potent in vitro and in vivo activities against drug-resistant Streptococcus pneumoniae. Among the fluoroquinolone compounds, zabofloxacin showed the most potent in vitro activity against clinical isolates of penicillin-sensitive S. pneumoniae (minimum inhibitory concentration, MIC90: 0.03 mg/L) and penicillin-resistant S. pneumoniae (MIC90: 0.03 mg/L). Against quinolone-resistant S. pneumoniae, zabofloxacin (MIC90: 1 mg/L) was more active than ciprofloxacin, sparfloxacin, and moxifloxacin; however, its activity was the same as that of gemifloxacin. The in vivo activity of zabofloxacin was most potent among the quinolone compounds tested against the systemic infection and respiratory tract infection models in mice.