Antimicrobial resistance represents a growing threat to global public health. The indiscriminate use of antibiotics has accelerated the emergence of resistant strains, reducing the therapeutic effectiveness of currently available drugs, fluoroquinolones being no exception. In this context, the design of new antimicrobials remains a significant challenge. This study evaluated, using in silico tools, the binding affinity of four novel fluoroquinolone derivatives against the DNA gyrase of six bacterial species, using moxifloxacin as the reference compound. Target protein sequences were retrieved from the Protein Data Bank and GenBank and subsequently modeled using SwissModel, I-TASSER, Phyre, and AlphaFold. The generated structures were assessed with MolProbity, and those with the best scores were selected for molecular docking. Proteins were prepared using Chimera 1.18 and AutoDockTools 1.5.7. The active site was identified with Discovery Studio 2024. Ligands were built in ZINC, prepared using Open Babel v3.1.1.60, and docked with AutoDock Vina v1.2.3.57. Docking validation was performed with DockRMSD. Residues SER83 (within the QRDR region), ARG121, and PTR122 (outside this region) were involved in ligand-enzyme interactions. Molecule C showed the highest binding affinity across all bacterial species, outperforming the control, while molecules A, B, and D displayed similar values to the reference compound. These findings suggest that molecule C exhibits a favorable profile as a potential antimicrobial agent against resistant strains.