Bacterial resistance represents a worldwide emergency threatening the efficacy of all available antibiotics. Among the several resistance mechanisms developed by bacteria, beta-lactamases enzymes (BLs), able to inactivate most beta-lactam core antibiotics, represent a key target to block prolonging antibiotics half-life. Several approaches aimed at inhibiting BLs have been so far undertaken, mainly involving beta-lactam like or covalent inhibitors. Applying a structure based de novo design approach, we recently discovered a novel, non-covalent and competitive inhibitor of AmpC beta lactamases: it has a Ki of 1 µM, a ligand efficiency of 0.38 kcal ∙ mol–1 and lead-like physical properties. Moreover, it reverts resistance to ceftazidime in bacterial pathogens expressing AmpC and does not up-regulate beta-lactamase expression in cell culture. Its features make it a good candidate for chemical optimization: starting from its crystallographic complex with AmpC, eleven analogues were designed to complement additional AmpC sites, then synthesized and tested against clinical resistant pathogens. While the new inhibitors maintain similar in vitro activity as the starting lead, some of them exert a higher potency in in vivo tests, showing improved synergic potency with ceftazidime in resistant clinically isolated strains.