The constant menace of infections caused by many microorganisms is one of the significant concerns to humankind. Also, implant-associated biofilms are prevalent in the healthcare sector, which opens up the need for various protective armories against diseases, including antibacterial coatings. In the current context, developing an appropriate antibacterial coating for the materials utilized in the health sector has significance. Synthesis of copper oxide (CuO) nanoparticles (NPs) by the chemical precipitation method and capping of CuO with carboxymethyl chitosan (CMC) provides outstanding antibacterial action. Natural rubber is widely used for various biomedical purposes, regardless its dormancy against most pathogens. An economical and practical method has been developed to transform the Natural Rubber Latex (NRL) film surface to be sufficiently antibacterial by CuO/CMC lamination. This paper discusses the antibiofilm activity of the rubber films against Escherichia coli (MTCC No. 729) and Staphylococcus aureus (MTCC No. 902), and the non-cytotoxicity of the NRL films after CuO/CMC lamination. Hence this surface modification over the NRL film provides excellent prospects for antibacterial polymer materials.