Glioblastoma multiforme (GBM) is neoplasm which is resistant to all currently used treatment modalities including surgery, radiation therapy and chemotherapy. Photodynamic therapy (PDT) has been suggested as a novel therapeutical approach to the treatment of malignant gliomas. Here, we attempted to enhance hypericin-induced photocytotoxicity and apoptosis by diazepam, a non-selective ligand of peripheral benzodiazepine receptors (PBR) which seem to play an important role in apoptosis regulation. For the study, we used U-87 MG and U373 MG glioma cell lines and primary cultures of GBM cells prepared from peroperatively obtained tumor specimens. The patients included 7 histologically confirmed GBMs. Colorimetric MTT assay was employed to study the photocytotoxic effects of hypericin and diazepam. Flow cytometry was used to detect apoptosis and assess the proapoptotic effects of diazepam. We found that hypericin upon photoactivation exerts strong cytotoxic effects against U-87 MG and U373 MG cells as well as primary GBM cell cultures. No cytotoxic effect of hypericin was observed under dark conditions. Diazepam inhibited cell growth in U-87 MG cells and primary cultures whereas proliferation of U373 MG cells remained unaffected. When hypericin was combined with diazepam, photocytotoxicity was increased in U-87 MG cells and primary cultures unlike U373 MG cells. Flow cytometric analysis revealed photoactivated hypericin-induced apoptosis in both cell lines. Apoptosis was significantly enhanced by diazepam in U-87 MG cells. However, no such effect was observed in U373 MG cells. In the present study, we showed that photocytotoxic effect of hypericin in glioma cells can be potentiated by diazepam. This effect is underlied by the ability of diazepam to facilitate hypericin-induced apoptosis. This work provides support to performe clinical studies involving diazepam in the antiglioma treatment regimens as an apoptosis-modulating agent.