Here we report a theoretical-experimental study of 4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine derivatives that act as inhibitors of bacterial DNA gyrase B (GyrB). A comprehensive analysis of the various molecular interactions that stabilize the molecular complexes was performed using combined theoretical techniques. Our results showed that QTAIM (Quantum Theory of Atoms In Molecules) calculations are a very useful tool for quantitatively describing the molecular interactions to determine which amino acids interact with the ligands. Moreover, our simulations have shown that this approach not only provides a detailed description of the different affinities of the ligands, but also has predictive power for compounds that have not yet been synthesized. In fact, we have synthesized and tested three new DNA gyrase inhibitors, two of which exhibit significant inhibitory activity. One of them binds spatially differently from known GyrB inhibitors because its 3-oxopropanoic acid moiety is oriented toward a previously unexplored subsite of the binding pocket.