Application of nanofibers for the purpose of tissue mimicking and regeneration has become widespread in the field of biomedicine. In this study, polyethersulfone (PES) electrospun nanofibrous membranes were fabricated, modified, and loaded with unrestricted somatic stem cells (USSC) to mimic the natural structure of bone. Untreated PES, plasma-treated PES, and collagen-grafted PES (COL-PES) nanofibers were characterized via Brunauer-Emmett-Teller method, attenuated total reflection Fourier transform infrared, contact angle measurements, and scanning electron microscopy. Their capacity to support proliferation, infiltration, and osteogenic differentiation of USSC was investigated using MTT assay, real-time reverse transcriptase-polymerase chain reaction, histologic staining, alkaline phosphatase activity, and calcium content assay. All the scaffolds had nanofibrous and highly porous structure with large surface area. After surface treatments, hydrophilicity of scaffolds increased intensively and their biocompatibility improved. During osteogenic differentiation of stem cells, alkaline phosphatase activity and calcium content exhibited the highest level in cells on COL-PES. Real-time reverse transcriptase-polymerase chain reaction showed significant difference between the expression levels of osteoblast-related genes on COL-PES compared to other scaffolds. Excellent infiltration of USSC was observed in nanofibrous membranes especially COL-PES. It can be concluded that COL-PES nanofibrous scaffold has potential for bone grafting because of its three-dimensional structure and bioactivity which enhance proliferation, differentiation, and infiltration of USSC.