Silver-functionalized reduced graphene oxide (Ag-rGO) nanosheets were prepared by single chemical and thermal processes, with very low concentration of silver. The resulting carbon framework consists of reduced graphene oxide (rGO) sheets or 3D networks, decorated with anchored silver nanoparticles. The Ag-rGO nanosheets were dispersed into a polymer matrix and the composites evaluated for use as biological scaffolds. The rGO material in poly(dimethylsiloxane) (PDMS) has been tested for antimicrobial activity against Gram-positive Staphylococcus aureus (S. Aureus) bacteria, after exposure times of 24 and 120 hours, as well as in the determination of cell viability on cultures of fibroblast cells (NIH/3T3). Using 1 mL of Ag-rGO in PDMS the antibacterial effectiveness against Staphylococcus aureus was limited, showing an increased amount of Colony Forming Units (CFU), after 24 hours of contact. In the cell viability assay, after 48 hours of contact, the group of 1 mL of Ag-rGO with PDMS was the only group that increased cell viability when compared to the control group. In this context, it is believed these behaviors are due to the increase in cell adhesion capacity promoted by the rGO. Thus, the Ag-rGO/PDMS hybrid nanocomposite films can be used as scaffolds for tissue engineering, as they limit antimicrobial activity.