Bacterial DNA topoisomerases are essential for bacterial growth and are attractive important targets for developing antibacterial drugs. Consequently, different potent inhibitors targeting bacterial topoisomerases have been developed. However, developing potent broad-spectrum inhibitors against both Gram-positive (G+) and Gram-negative (G-) bacteria have been proven to be 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 demonstrate that the inhibitor forms a tight complex with ParEs and the complexes adopt similar structural conformations as compared to free ParEs in solution. Further chemical shift perturbation experiment and NOE analysis indicated that there are four regions in ParE that are important for inhibitor binding, namely the α2, loop between β2 and α3, β2 and β6 strands. Surface Plasmon resonance (SPR) showed that this inhibitor binds to sParE with a higher KD than pParE. Point mutations in α2 of ParE such as A52S (sParE) affected its binding affinity with the inhibitor. Taken together, the results provide better understanding of developing broad spectrum antibacterial agents.