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In this study, Cu 2 O (copper oxide) nanoparticles were synthesized by using mechanical milling and tested for antibacterial properties. The raw material for copper oxide powder (macroparticles) was refined in size using an instrument of shaker mill with a 1: 1 ball and powder ratio and a time variation of up to 16 hours. X-Ray Diffraction (XRD) diffraction data showed changes in crystallinity and size depending on the grinding time from the calculation of the crystallite size. The results of microstructure observation using an electronic microscope (Scanning Electron Microscope, SEM) also showed changes in particle size correlated with the length of the milling process. Antibacterial activity was carried out on the Vibrio eltor, Bacillus subtilis, Staphylococcus aureus, Candida tropicalis, and Saccharomycetes cerevisiae with the effect of particle size on antibacterial activity is discussed.