In the last few years, graphene oxide (GO) has gained considerable importance in scaffold preparation for tissue engineering due to the presence of functional groups that allow the interaction between the extracellular matrix and the components of the cellular membrane. The interaction between GO and chitosan (CS) can not only improve the biomechanical properties of the scaffold but also generate a synergistic effect, facilitating tissue recovery. In vivo studies on GO are scarce; therefore, biocompatibility tests on CS-GO scaffolds and bone regeneration experiments on critical size defects were carried out on Wistar rats. Scaffolds made of CS, CS-GO 0.5%, and CS-GO 1% were prepared and implanted on Wistar rats cranial bones for three months. Scaffold samples were analyzed through histochemistry and scanning electron microscopy. The analysis performed showed reabsorption of the material by phagocytic activity and new bone formation. The CS-GO 0.5% formulation gave the best performance in bone regeneration, with excellent biocompatibility. These results show the potential of this compound for tissue regeneration opening and medical applications.