Pazufloxacin (T-3761) is a fluoroquinolone antibiotic used for antibacterial applications. This novel quinolone derivative exhibits robust and broad-spectrum antibacterial efficacy against systemic, pulmonary, and urinary tract infections in mice, effectively targeting both gram-positive and gram-negative bacteria, including quinolone-resistant strains such as Serratia marcescens and Pseudomonas aeruginosa. Compared to antibiotics like ofloxacin, ciprofloxacin, norfloxacin, and tosufloxacin, Pazufloxacin demonstrates comparable or superior efficacy across various infection models. While slightly less effective than tosufloxacin against certain gram-positive infections, it remains potent against methicillin-resistant Staphylococcus aureus. Its broad-spectrum activity is supported by minimum inhibitory concentrations (MICs) between 0.39 and 6.25 micrograms/ml for Staphylococcus aureus and Clostridium species, with minimum bactericidal concentrations (MBCs) close to the MICs. Additionally, its 50% inhibitory concentrations for DNA gyrases from E. coli and P. aeruginosa are 0.88 and 1.9 micrograms/ml respectively, highlighting its potent antibacterial mechanism of action.