Bacterial DNA topoisomerases are essential for bacterial growth and are attractive, important targets for developing antibacterial drugs. Consequently, different potent inhibitors that target bacterial topoisomerases have been developed. However, the development of potent broad-spectrum inhibitors against both Gram-positive (G+) and Gram-negative (G-) bacteria has proven challenging. In this study, we carried out biophysical studies to better understand the molecular interactions between a potent bis-pyridylurea inhibitor and the active domains of the E-subunits of topoisomerase IV (ParE) from a G+ strain (Streptococcus pneumoniae (sParE)) and a G- strain (Pseudomonas aeruginosa (pParE)). NMR results demonstrated that the inhibitor forms a tight complex with ParEs and the resulting complexes adopt structural conformations similar to those observed for free ParEs in solution. Further chemical-shift perturbation experiments and NOE analyses indicated that there are four regions in ParE that are important for inhibitor binding, namely, a2, the loop between ?2 and a3, and the ?2 and ?6 strands. Surface plasmon resonance showed that this inhibitor binds to sParE with a higher KD than pParE. Point mutations in a2 of ParE, such as A52S (sParE), affected its binding affinity with the inhibitor. Taken together, these results provide a better understanding of the development of broad-spectrum antibacterial agents.