Gyrase and topoisomerase IV, enzymes that play critical roles during DNA replication, are the targets of fluoroquinolones and other antibacterials. Gyrase removes positive supercoils that accumulate ahead of replication forks, while topoisomerase IV untangles daughter chromosomes. Although topoisomerase IV is an essential enzyme in most bacteria, some species, including Mycobacterium tuberculosis and Mycobacteroides abscessus, encode only gyrase. In these species, gyrase is the sole target of fluoroquinolones and is believed to assume the cellular functions of both type II topoisomerases. Although fluoroquinolones and emerging antibacterials such as spiropyrimidinetriones induce gyrase-mediated DNA cleavage, there is evidence that inhibition of gyrase function also plays a role in drug-induced cell death under some circumstances. Therefore, we examined the effects of moxifloxacin and ciprofloxacin (fluoroquinolones) and zoliflodacin (spiropyrimidinetrione) on the three catalytic activities presumably carried out by gyrase in mycobacteria: decatenation of tangled DNA, negative supercoiling of relaxed DNA, and relaxation of positive supercoils. Under all circumstances, lower concentrations of antibacterials were required to inhibit intermolecular DNA decatenation as compared to the intramolecular DNA relaxation or supercoiling functions of gyrase. Differences in drug potency could not be attributed solely to rates of individual reactions or the DNA substrates utilized. Rather, results suggest that the potency of gyrase inhibition by interfacial antibacterials is modulated by the topological state of the DNA and its specific interactions with gyrase. Whereas most studies focus on DNA cleavage induced by gyrase-targeted antibacterials, this study provides mechanistic insights into how antibacterials rob replicating cells of essential gyrase functions.