The development of nanoscale drug delivery systems, which can mediate efficient tumor targeting together with high cellular internalization, is crucial for glioma treatment. The combination of therapeutic agents in nanoparticles provides synergistic effects and allows further surface modifications with targeting ligands for specific glioma therapy. To achieve this goal, both doxorubicin and curcumin were encapsulated in polyethylene glycolated niosomes (PEGNIO). The surface of co-drug loaded PEGNIO was modified with tumor homing and penetrating peptide (tLyp-1). Physicochemical properties were determined via dynamic light scattering (DLS) and spectral analysis. Moreover, flow cytometry studies were performed to examine the specific cellular uptake of the tLyp-1 targeted niosomal formulation. In vitro cytotoxicity and inhibition of tumor-like spheroids growth were investigated on human glioblastoma (U87) and human mesenchymal stem cells (hMSC) cells. The results clearly indicated that the strategy by co-administration of doxorubicin and curcumin with tLyp-1 functionalized niosomes could significantly improve anti-glioma treatment.