The rise in quinolone resistance is threatening the clinical use of this important class of broad-spectrum antibacterials. Quinolones kill bacteria by increasing the level of DNA strand breaks generated by the type II topoisomerases gyrase and topoisomerase IV. Most commonly, resistance is caused by mutations in the serine and acidic amino acid residues that anchor a water-metal ion bridge that facilitates quinolone-enzyme interactions. Although other mutations in gyrase and topoisomerase IV have been reported in quinolone-resistant strains, little is known regarding their contributions to cellular quinolone resistance. To address this issue, we characterized the effects of the V96A mutation in the A subunit of Bacillus anthracis topoisomerase IV on quinolone activity. The results indicate that this mutation causes an ∼3-fold decrease in quinolone potency and reduces the stability of covalent topoisomerase IV-cleaved DNA complexes. However, based on metal ion usage, the V96A mutation does not disrupt the function of the water-metal ion bridge. A similar level of resistance to quinazolinediones (which do not use the bridge) was seen. V96A is the first topoisomerase IV mutation distal to the water-metal ion bridge demonstrated to decrease quinolone activity. It also represents the first A subunit mutation reported to cause resistance to quinazolinediones. This cross-resistance suggests that the V96A change has a global effect on the structure of the drug-binding pocket of topoisomerase IV.