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Today, tissue regeneration is one of the greatest challenges in the field of medicine, since it represents hope after accidents or illnesses. Tissue engineering is the science based on improving or restoring tissues and organs. In this work, five formulations of chitosan/poly(vinyl alcohol)/graphene oxide (CS/PVA/GO) nanocomposites were studied for the development of biodegradable films with potential biomedical applications. The characterization of the films consisted of Fourier-transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). The antibacterial activity was evaluated in vitro against Gram-positive bacteria Bacillus cereus and Staphylococcus aureus and Gram-negative Salmonella spp. and Escherichia coli, by contact of the film above inoculum bacterial in Müeller–Hinton agar. On the other hand, in vivo tests in which the material implanted in the subcutaneous tissue of Wistar rats demonstrated that the formulation CS/PVA/GO (14.25:85:0.75) was the best antibacterial film with adequate degradation in vivo. All together, these results indicate the potential of the films using nanocomposites of CS/PVA/GO in tissue engineering and cell regeneration.