This research article investigates the impact of three input variables, including Cu Composition (%), Heating Temperature (C), and Milling Time (h), on the final production of copper-tin alloy nanoparticles for the first time. The study uses design of experiments techniques and measures three output responses, including Particle Size (nm), Optical Density (ppm), and Number of Colonies. The research identifies the presence of new Cu3Sn phases in the final structure of nanoparticles. The results demonstrate that all three input factors have a significant impact on nanoparticle production, with mechanical alloying effectively producing nanoparticle powders up to 15 nanometers in size. The study reveals that increasing the percentage of copper in the final alloy leads to stronger antibacterial properties, as demonstrated by increased optical density and decreased colony counts. This work provides valuable insights into the antibacterial properties of copper-tin alloy nanoparticles and the influence of input variables on their structure and properties.