Nanofiber scaffolds of polyvinyl alcohol, silk fibroin from Bombyx mori cocoons, and silver nanoparticles were developed as a substrate for MG-63 growth. The fiber morphology, mechanical properties, thermal degradation, chemical composition, and water contact angle were investigated. In vitro tests were performed by the cell viability MTS test of MG-63 cells on electrospun PVA scaffolds, mineralization was analyzed by alizarin red, and the alkaline phosphatase (ALP) assay was evaluated. At higher PVA concentrations, Young’s modulus (E) increased. The addition of fibroin and silver nanoparticles improved the thermal stability of PVA scaffolds. FTIR spectra indicated characteristic absorption peaks related to the chemical structures of PVA, fibroin, and Ag-NPs, demonstrating good interactions between them. The contact angle of the PVA scaffolds decreased with the incorporation of fibroin and showed hydrophilic characteristics. In all concentrations, MG-63 cells on PVA/fibroin/Ag-NPs scaffolds had higher cell viability than PVA pristine. On day ten of culture, PVA18/SF/Ag-NPs showed the highest mineralization, observed by the alizarin red test. PVA10/SF/Ag-NPs presented the highest alkaline phosphatase activity after an incubation time of 37 h. The achievements indicate the potential of the nanofibers of PVA18/SF/Ag-NPs as a possible substitute for bone tissue engineering (BTE).