In this study, the effects of ethanol treatment on the mechanical and biological characteristics of the nanofibrous silk fibroin (NSF) scaffolds were evaluated. The results obtained from the mechanical tests confirmed that ethanol treatment significantly enhanced the physical properties of the scaffolds through the formation of a ß-sheet structure. It was shown that the ethanol treatment increased the mechanical property and cell viability, while decreased the porosity of the randomly arranged uniform nanofibers. The ultimate tensile strength for the NSF and ethanol-treated NSF (ET-NSF) scaffolds were 0.76 and 1.33 MPa, respectively. In addition, the ethanol treatment positively affected the proliferation rate of rat bone-marrow stromal cells (rBMSCs) without any detectable cytotoxicity. All the results obtained from this study strongly indicated the efficacy of ethanol treatment in enhancement of mechanical and biological characteristics of silk fibroin nanofibrous scaffolds.