BACKGROUND: Tosufloxacin is one of the fluoroquinolones with higher antibacterial activity against many organisms than other fluoroquinolones developed previously. However, the antimicrobial susceptibilities of tosufloxacin against uropathogens are not well known. The objective of this study was to compare minimum inhibitory concentrations (MICs) of tosufloxacin with those of levofloxacin and ofloxacin for uropathogens cultured from the urine of female outpatients with acute uncomplicated cystitis. MATERIALS AND METHODS: We collected and cultured urine samples from four metropolitan hospitals between August 2006 and November 2006. The MICs of tosufloxacin, levofloxacin and ofloxacin were determined for 89 isolates using an agar dilution method. RESULTS: The most prevalent causative organism was Escherichia coli (70.8%). The MIC distribution of tosufloxacin and levofloxacin showed a bimodal distribution for E. coli. The two modes for E. coli were 0.03 and 32 microgram/mL for tosufloxacin and 0.03 and 16 microgram/mL for levofloxacin. However, ofloxacin had one more peak at MIC 0.5 microgram/mL. MIC50s of tosufloxacin, levofloxacin and ofloxacin for E. coli were 0.03 microgram/mL, 0.06 microgram/mL and 0.125 microgram/mL, respectively. Tosufloxacin, the newer fluoroquinolone, was more active than levofloxacin and ofloxacin against both E. coli and Gram-positive bacteria. CONCLUSION: Of the studied fluoroquinolones tosufloxacin had the lowest MICs against uropathogens and could be considered a promising alternative fluoroquinolone agent for the treatment of urinary tract infection.