The in vitro activity of fleroxacin was determined by broth microdilution against 2,079 recent bacterial isolates and compared to the activities of ciprofloxacin, ofloxacin, lomefloxacin, cefaclor, cefuroxime, cefixime, ceftriaxone, amoxicillin/clavulanate, trimethoprim/sulfamethoxazole (TMP-SMX), and, as appropriate, erythromycin and oxacillin. Most Enterobacteriaceae were inhibited by the quinolones at a concentration of < or = 1 microgram/ml; MIC90s of fleroxacin, ciprofloxacin, ofloxacin, and lomefloxacin were 0.25, 0.5, 1 and 1 micrograms/ml, respectively. Fleroxacin was 2-fold more active than ciprofloxacin against Providencia stuartii and Serratia marcescens. Aside from the quinolones, ceftriaxone and TMP-SMX were the most active antibiotics against the Enterobacteriaceae, with MIC90s of 8 and 16 micrograms/ml, respectively. Ciprofloxacin was more active against Pseudomonas aeruginosa than the other quinolones, while fleroxacin was more active against Stenotrophomonas maltophilia: 17.7, 11.2, 20.0, and 22.4% of P. aeruginosa were resistant to fleroxacin, ciprofloxacin, ofloxacin, and lomefloxacin, respectively. Moraxella catarrhalis and Haemophilus influenzae were uniformally susceptible to all antibiotics tested, as were the majority of oxacillin-susceptible staphylococci. The MIC90s of the quinolones and of the beta-lactam antibiotics for oxacillin-resistant staphylococci were 8- to 256-fold higher than for oxacillin-susceptible staphylococci. The beta-lactam antibiotics, TMP-SMX, and erythromycin were more active than the quinolones against streptococci; all antibiotics were poorly active against enterococci. Fleroxacin is active against a broad range of gram-negative bacilli and against oxacillin-susceptible staphylococci and should prove useful for such infections. However, its use cannot be recommended for infections due to oxacillin-resistant staphylococci, streptococci, or enterococci.