Glioblastoma multiforme (GBM) is considered to be one of the most serious version of primary malignant tumors. Temozolomide (TMZ), an anti-cancer drug, is the most common chemotherapeutic agent used for patients suffering from GBM. However, due to its inherent instability, short biological half-life, and dose-limiting characteristics, alternatives to TMZ have been sought. In this study, the TMZ-loaded PLGA nanoparticles were prepared by employing the emulsion solvent evaporation technique. The prepared TMZ-PLGA-NPs were characterized using FT-IR, zeta potential analyses, XRD pattern, particle size estimation, TEM, and FE-SEM observations. The virotherapy, being safe, selective, and effective in combating cancer, was employed, and TMZ-PLGA-NPs and oncolytic Newcastle Disease Virus (NDV) were co-administered for the purpose. An AMHA1-attenuated strain of NDV was propagated in chicken embryos, and the virus was titrated in Vero-slammed cells to determine the infective dose. The in vitro cytotoxic effects of the TMZ, NDV, and the TMZ-PLGA-NPs against the human glioblastoma cancer cell line, AMGM5, and the normal cell line of rat embryo fibroblasts (REFs) were evaluated. The synergistic effects of the nano-formulation and viral strain combined therapy was observed on the cell lines in MTT viability assays, together with the Chou-Talalay tests. The outcomes of the in vitro investigation revealed that the drug combinations of NDV and TMZ, as well as NDV and TMZ-PLGA-NPs exerted the synergistic enhancements of the antitumor activity on the AMGM5 cell lines. The effectiveness of both the mono, and combined treatments on the capability of AMGM5 cells to form colonies were also examined with crystal violet dyeing tests. The morphological features, and apoptotic reactions of the treated cells were investigated by utilizing the phase-contrast inverted microscopic examinations, and acridine orange/propidium iodide double-staining tests. Based on the current findings, the potential for the use of TMZ and NDV as part of a combination treatment of GBM is significant, and may work for patients suffering from GBM.