Cow milk-derived carbon dots (CMCDs) were prepared by hydrothermal treatment of cow milk, and the as-prepared CMCDs were further extracted by ethyl acetate to obtain amphiphilic CMCDs (ACM-CDs). Using the ACMCDs both as a reducing agent and a template, the ACMCDs-supported silver nanoparticles (ACMCD-Ag nanocomposites) were prepared, which showed good biocidal effect on both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacterias. After that, a novel ACMCD-Ag/polymethylmethacrylate nanocomposite antibacterial film was fabricated by solvent casting method. Due to the excellent antibacterial, light admitting, and flexible properties, the nanocomposite antibacterial film is considered to be of great potential in applications. (C) 2014 Elsevier B.V. All rights reserved.