Synthesis, characterization, and antibacterial properties of Cu and Cu/Ni nanoparticles loaded on NaP zeolite against Bacillus subtilis and Escherichia coli were investigated. Synthesis of Cu and Ni nanoparticles of the NaP substrate was carried out by conventional and sonochemical methods in ethylene glycol medium. A Box-Behnken design of experiment was employed to analyze the influence of nanoparticle loading, Cu content of nanoparticles, and concentration of metallic salts solution on the antibacterial activity of the synthesized product. The synthesized samples provided an about one-third higher activity against B. subtilis compared to E. coli. Ultrasonic irradiation significantly improved the antibacterial activity against E. coli and B. subtilis. Time-and concentration-dependent bacterial tests of the synthesized sample under optimum conditions were performed with a central composite design of experiment.