The median survival of patients with glioblastoma multiforme (GBM) remains poor. Innovative immunotherapies with dendritic cell (DC) vaccination might be combined with standard temozolomide (TMZ) treatment. Here, we evaluated the influence of TMZ on the phenotype and function of DCs and CD8+ T cells. DCs were generated from the peripheral blood of healthy volunteers (HVs) and GBM patients. DCs were analyzed by light microscopy and flow cytometry. Phagocytic activity was tested by FITC-dextran engulfment. Mixed lymphocyte peptide cultures were followed by enzyme-linked immunospot (ELISPOT) and flow cytometry assays. TMZ was added to DC and T cell cultures at concentrations up to 500 µM. Mature DCs were generated from HVs and GBM patients. Cells displayed a typical DC morphology and a mature DC phenotype. Expression of CD209 was even higher in DCs generated from patients under therapy than from HVs (75.2 vs. 51.1%). In contrast, CD40 (1.1 vs. 13.5%) and BDCA4 (26.5 vs. 52.9%) were lower expressed in GBM patients at time of diagnosis. Immature DCs showed high phagocytic activity. Addition of TMZ at concentrations up to 50 µM did neither impair the phenotype nor the function of DCs. In ELISPOT and flow cytometry assays, no impairment of CD8+ T cell responses to viral antigens could be observed. Taken together, TMZ does not impair the function of either DCs or the CD8+ T cells.