<jats:title>Abstract</jats:title><jats:p>Infectious pathogens can be transmitted through textiles. Therefore, additional efforts are needed to develop functional fabrics containing antimicrobial substances to prevent the growth of antibiotic-resistant bacteria and their biofilms. Here, we developed a cotton fabric coated with reduced graphene oxide (rGO) and copper nanoparticles (Cu NPs), which possessed hydrophobic, antimicrobial, and anti-biofilm properties. Once the graphene oxide was dip-coated on a cellulose cotton fabric, Cu NPs were synthesized using a chemical reduction method to fabricate an rGO/Cu fabric, which was analyzed through FE-SEM, EDS, and ICP-MS. The results of our colony-forming unit assays indicated that the rGO/Cu fabric possessed high antibacterial and anti-biofilm properties against <jats:italic>Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis</jats:italic>, <jats:italic>Corynebacterium xerosis,</jats:italic> and <jats:italic>Micrococcus luteus</jats:italic>. Particularly, the fabric could inhibit the growth of <jats:italic>E. coli, C. xerosis,</jats:italic> and <jats:italic>M. luteus</jats:italic> with a 99% efficiency. Furthermore, our findings confirmed that the same concentrations of rGO/Cu had no cytotoxic effects against CCD-986Sk and Human Dermal Fibroblast (HDF), human skin cells, and NIH/3T3, a mouse skin cell. The developed rGO/Cu fabric thus exhibited promising applicability as a cotton material that can maintain hygienic conditions by preventing the propagation of various bacteria and sufficiently suppressing biofilm formation while also being harmless to the human body.</jats:p>