Reduced graphene oxide (rGO) has been widely used to fabricate electronics, sensors, photodetectors,and in other applications. However, the antibacterial performance of pristine rGO is relatively weak. The application of rGO in biomedical devices, smart food packaging, and water desalination membranes requires further improvement of rGO’s antibacterial abilities. Copper(I) oxide (Cu₂O) is an effective antibacterial agent, which denatures protein and enhances the permeability of cell membranes. In this work, we report a simple method of synthesizing a highly antibacterial rGO/Cu₂O nanocomposite from cellulose acetate, a derivative of abundant natural cellulose. The synthesized rGO/Cu₂O nanocomposite was thoroughly characterized by Raman spectroscopy, X-ray powder diffraction (XRD), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), and scanning transmission electron microscopy (STEM). Then, the antibacterial abilities of rGO/Cu₂O nanocomposite were evaluated and a bactericidal mechanism was revealed from the molecular biology perspective. Results indicate that our synthesized rGO/Cu₂O nanocomposite owns strong antibacterial activity, mainly stemming from the uniformly incorporated Cu₂O nanocrystals with a lateral size of 5–40 nm.