Introduction: Enterobacter cloacae is a pathogenic microorganism with the ability to produce betalactamase enzymes, which makes them resistant to betalactamic antibiotics. Additionally, the limited activity of enzymatic inhibitors has been identified, and, therefore, the design of new drugs and the promotion of their rational use are the only possibilities to overcome this problem.Objective: The aim of this research was to evaluate the effect of dihydroxy-phenyl-propenone on a clinical isolate of E. cloacae, as well as its activity on a betalactamase isolated from this resistant microorganism in order to contribute to the search for new betalactamase inhibitors.Materials and methods: Dihydroxy-phenyl-propenone chalcone was synthesized and evaluated on a clinical isolate of E. cloacae to determine the minimum inhibitory concentration by broth microdilution; once the betalactamase enzyme was purified by affinity chromatography, a spectrophotometric analysis was done to evaluate its kinetic activity.Results: The minimum inhibitory concentration value of dihydroxy–phenyl-propenone on E. cloacae was 35 μg/ml; the recovery percentage of the betalactamase from the microorganism was 31.75% and the kinetic parameters were Vmax=1.7 x 10-3 μM/min and KM= 2330 μM, which show an important inhibitory activity.Conclusion: Dihydroxy-phenyl-propenone has shown inhibitory activity on betalactamase enzymes and the ability to protect the chemical integrity of betalactamic antibiotics; this synergistic effect turns it into a promising compound in the search for new alternatives to overcome bacterial resistance.