<p>Delafloxacin is a potent anionic fluoroquinolone approved for the treatment of respiratory infections that acts by trapping the DNA cleavage complexes of bacterial topoisomerase IV and gyrase. Its N-1-pyridinyl-, C-7-azetidinyl- and C-8-chlorine substituents confer enhanced antibiotic activity against bacteria resistant to other fluoroquinolones, but its mode of action is unclear. Here we present the X-ray crystal structures of a delafloxacin-DNA cleavage complex obtained by co-crystallization with <i>Streptococcus pneumoniae</i> topo IV using a graphene nucleant and solved at 2.0 and 2.4 Å resolution. The two Mg²⁺-chelated delafloxacin molecules intercalated at the DNA cleavage site are bound in an unusual conformation involving interacting out-of-plane N-1-aromatic- and C-8-chlorine- substituents. The unprecedented resolution allows comprehensive imaging of water-metal ion links integrating enzyme and DNA through drug-bound and active-site Mg²⁺ ions plus the discovery of enzyme-bound K⁺ ions. Our studies on delafloxacin action suggest that intrinsic target affinity contributes to its activity against quinolone-resistant bacteria.</p>