This study investigates the effect of mechanical milling time on the structural properties and bioactivity of metal oxide powders composed of CuO, MgO, and ZnO. Nanocomposites (NPCs) were synthesized by milling at 0 h, 5 h, and 10 h to evaluate their potential in biomedical and food packaging applications. The samples were characterized using X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Spectroscopy (EDS), and Fourier Transform Infrared Spectroscopy (FT-IR). XRD analysis revealed a reduction in crystallite size from 23.09 nm to 21.67 nm with increasing milling time. SEM-EDS analysis showed a homogeneous dispersion of elements in the 5 h sample. FT-IR confirmed the formation of new bonds between the metal oxides, suggesting changes in the material's structure. These structural modifications directly impact the material's bioactivity. Antibacterial activity tests demonstrated that NPCs exhibit greater efficacy against Staphylococcus aureus, with the 5 h sample being the most effective, achieving 46.7 % inhibition at 3 mg/mL. The 10 h sample showed similar efficacy (43.3 %). Gramnegative bacteria, such as Escherichia coli and Salmonella spp., exhibited minimal inhibition, likely due to the resistance mechanisms of their cell walls, which limit the effectiveness of antibacterial agents. The relationship between structural properties and bioactivity was clear, as the reduction in particle size and improved dispersion of elements enhanced antibacterial activity. Cytotoxicity tests showed that NPCs were non-toxic to erythrocytes, with cell viabilities above 75 %, meeting biocompatibility standards established by ISO 10993-5. Nanocomposites synthesized after 5 h of milling (NPC 5 h) proved to be the most effective in terms of biocompatibility, with the highest cell viability recorded at 97.40 % at a concentration of 0.75 mg/mL. Even at a concentration of 3.00 mg/mL, NPC 5 h maintained cell viability above 74.80 %, highlighting its potential as a biocompatible material. The optimal milling time of 5 h achieved a balance between antibacterial activity and biocompatibility, representing this study's novel contribution by identifying the appropriate milling time to enhance both antibacterial efficacy and biocompatibility.