© 2015 American Chemical Society. CP-115,955 is a quinolone with a 4-hydroxyphenyl at C7 that displays high activity against both bacterial and human type II topoisomerases. To determine the basis for quinolone cross-reactivity between bacterial and human enzymes, the activity of CP-115,955 and a series of related quinolones and quinazolinediones against Bacillus anthracis topoisomerase IV and human topoisomerase IIα was analyzed. Results indicate that the activity of CP-115,955 against the bacterial and human enzymes is mediated by different interactions. On the basis of the decreased activity of quinazolinediones against wild-type and resistant mutant topoisomerase IV and the low activity of quinolones against resistant mutant enzymes, it appears that the primary interaction of CP-115,955 with the bacterial system is mediated through the C3/C4 keto acid and the water-metal ion bridge. In contrast, the drug interacts with the human enzyme primarily through the C7 4-hydroxyphenyl ring and has no requirement for a substituent at C8 in order to attain high activity. Despite the fact that the human type II enzyme is unable to utilize the water-metal ion bridge, quinolones in the CP-115,955 series display higher activity against topoisomerase IIα in vitro and in cultured human cells than the corresponding quinazolinediones. Thus, quinolones may be a viable platform for the development of novel drugs with anticancer potential.