Optimization of nanofiber (NF) surface properties is critical to achieve an adequate cellular response. Here, the impact of conjugation of biomimetic aspartic acid (ASP) and glutamic acid (GLU) templated peptides with poly(lactic-co-glycolic acid) (PLGA) electrospun NF on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) was evaluated. Cold atmospheric plasma (CAP) was used to functionalize the NF surface and thus to mediate the conjugation. The influence of the CAP treatment following with peptide conjugation to the NF surface was assessed using water contact angle measurements, Fourier-Transform Infrared Spectroscopy (FTIR) and X-ray Photoelectron Spectroscopy (XPS). The effect of CAP treatment on morphology of NF was also checked using Scanning Electron Microscopy (SEM). Both the hydrophilicity of NF and the number of the carboxyl (-COOH) groups on the surface increased with respect to CAP treatment. Results demonstrated that CAP treatment significantly enhanced peptide conjugation on the surface of NF. Osteogenic differentiation results indicated that conjugating of biomimetic ASP templated peptides sharply increased alkaline phosphatase (ALP) activity, calcium content, and expression of key osteogenic markers of collagen type I (Col-I), osteocalcin (OC), and osteopontin (OP) compared to GLU conjugated (GLU-pNF) and CAP treated NF (pNF). It was further depicted that ASP sequences are the major fragments that influence the mineralization and osteogenic differentiation in non-collagenous proteins of bone extracellular matrix.