Quinolones that act equally against DNA gyrase and topoisomerase IV are a desirable modality to decrease the selection of resistant strains. We first determined by genetic and biochemical studies in Staphylococcus aureus that the primary target enzyme of WCK-1734, a new quinolone, was DNA gyrase. A single mutation in gyrase, but not topoisomerase IV, caused a two- to fourfold increase in the MIC. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that gyrase was more sensitive than topoisomerase IV to WCK-1734 (50% inhibitory concentration, 1.25 and 2.5 to 5.0 μg/ml, respectively; 50% stimulation of cleavage complex formation, 0.62 and 2.5 to 5.0 μg/ml, respectively). To test the effect of balanced activity of quinolones against the two target enzymes, we measured the frequency of selection of mutants with ciprofloxacin (which targets topoisomerase IV) and WCK-1734 alone and in combination. With the combination of ciprofloxacin and WCK-1734, each at its MIC, the ratio of frequency of mutants selected was significantly lower than that with each drug alone at two times their respective MICs. We further characterized resistant strains selected with the combination of ciprofloxacin and WCK-1734 and found evidence to suggest the existence of novel mutational mechanisms for low-level quinolone resistance. By use of a combination of differentially targeting quinolones, this study provides novel data in direct support of the paradigm for dual targeting of quinolone action and reduced development of resistance.