Glioblastoma multiforme is a malignant neoplasia with a median survival of less than two years and without satisfactory therapeutic options. The so-called glioblastoma stem cells escape the established radio-and che-motherapies and lead to tumor recurrence in most cases. The alkaloid Shikonin with its various anti stem cell properties and the interstitial photodynamic therapy with 5-aminolevulinic acid seem to be promising new options in the therapy of glioblastoma. In this study, in vitro investigations were performed to observe the in-fluence of Shikonin on viability, proliferation, induction of apoptosis and the capability of forming tumor spheres in U-87 MG and the primary glioblastoma cell line GB14. The combined effect with the chemotherapeutic temozolomide and photodynamic treatment on the mRNA expression of glioma specific stem cell markers and further examined intracellular protoporphyrin IX accumulation under Shikonin treatment was analyzed. Shi-konin effectively inhibited the capability of forming tumor spheres and enhanced temozolomide effectiveness in the reduction of proliferation and in the induction of apoptosis. Additionally, Shikonin increased the mRNA expression of the tumor suppressing Neurofibromatosis type 1 (NF1) gene and showed modulating effects on intracellular protoporphyrin IX.