This article describes the production of nanoparticles of Chilean natural zeolite, using three size reduction methods: Ball mill, microgrinding, and microfluidization. Morphological characterization of samples indicated an average diameter of 37.2 ± 15.8 nm of the zeolite particles. The size reduction and chemical treatments did not affect the morphology or integrity of the zeolite. An increase of the zeolite samples’ Si/Al ratio was observed after the acid treatment and was confirmed by SEM-EDX analysis. Moreover, the effectiveness of the copper salt ion exchange (Cu2+) to the zeolite nanoparticles was analyzed by SEM-EDX. XRD analysis indicated that clinoptilolite and mordenite are the main phases of Chilean natural zeolite, and the crystalline structure was not affected by the modification processes. The FTIR characterization showed the presence of chemical bonds of copper with the zeolite nanoparticle framework. The ion-exchanged zeolite nanoparticles were evaluated for antibacterial behavior by the disc diffusion method. Additionally, the minimum inhibitory concentration and minimum bactericidal concentration were obtained. Microbiological assays with copper-exchanged nanozeolites showed an antimicrobial activity with a bactericidal effect against Escherichia coli and Staphylococcus aureus, which are the primary pathogens of food and are also resistant to multiple drugs. In this study, a new application for natural nanozeolites is demonstrated, as the incorporated copper ions (Cu2+) in nanozeolites registered a productive antibacterial activity.