Background/Objectives: The study developed, characterized, and evaluated the toxicity, antibacterial and antibiofilm activity of quercetin encapsulated in chitosan-coated zein nanoparticles (QUER-ZNP-CH). Methods: QUER-ZNP-CH were prepared by the nanoprecipitation method and characterized by physicochemical analyses, stability (12 months), and release kinetics. Toxicity was evaluated through hemocompatibility and a Tenebrio molitor larval model. Antibacterial activity (MIC/MBC, CLSI) and antibiofilm potential (crystal violet assay) were tested against resistant Klebsiella pneumoniae strains. Results: The nanoparticles were prepared, and physicochemical analyses revealed chemical interactions, efficient encapsulation of the drug, and thermal stability. The formulations remained stable over 12 months, and the release kinetics demonstrated controlled release for 72 h. No hemotoxic profile was observed and there was 95% survival of Tenebrio molitor larvae after treatment with QUER-ZNP-CH. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of QUER-ZNP-CH revealed enhanced antibacterial activity of QUER, as indicated by a 32 to 64-fold reduction in the MIC and MBC values. The biofilm inhibition potential of QUER-ZNP-CH showed 60–100% inhibition and 25–95% eradication in concentrations from 0.12 to 62.5 μg/mL. Conclusions: Thus, this nanotechnology-based formulation suggests potential for the treatment of bacterial infections caused by multidrug-resistant K. pneumoniae strains.
