The purpose of this study was to investigate physical, mechanical, and osteogenic properties of silk fibroin (SF) nanofibers containing Urtica dioica L. (nettle) extract at different concentrations. In this respect, the successful incorporation of nettle in SF nanofibers was analyzed and then confirmed through Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The mean fiber diameter, water uptake, breaking strain, cellular attachment, and proliferation of the given nanofibers also increased as the nettle content was added, while this trend was opposite in terms of tensile strength and modulus. The in vitro release studies correspondingly demonstrated that the nettle release had been controlled according to Fickian diffusion and it was faster in the samples including more nettle. Furthermore, both ARS staining and real-time RT-PCR results suggested that nettle had enhanced the expression of both early and late markers of osteoblast differentiation in a dose-dependent manner.