We analyzed the relationship between resistance to nadifloxacin, a fluoroquinolone antimicrobial agent, and mutations in the quinolone resistance-determining regions (QRDRs) of the A subunit of DNA gyrase and topoisomerase IV, in 24 clinical isolates of Staphylococcus aureus. Seven known mutations were found in the QRDRs. The minimum inhibitory concentration (MIC) of nadifloxacin increased in a manner that was dependent on mutations in the A subunit of DNA gyrase, and did not appear to be related to mutations in the A subunit of topoisomerase IV. The type 9 mutant, which included four mutations, was highly resistant to ofloxacin, norfloxacin and sparfloxacin, but only moderately resistant to nadifloxacin (MIC, 12.5 μg/ml). One of the norfloxacin-resistant strains that expressed high levels of norA was not resistant to nadifloxacin. To the best of our knowledge, this is the first report describing a fluoroquinolone antimicrobial agent whose primary target is suggested to be DNA gyrase in S. aureus.