Glioblastoma is the most aggressive tumor of the central nervous system and is manifested by diffuse invasion of glioblastoma stem cells into the healthy tissue, chemoresistance and recurrence. Despite aggressive therapy, consisting of maximal surgical resection, radiotherapy and chemotherapy with temozolomide (Temodal®), life expectancy of patients with glioblastoma is typically less than 15 months. In general, natural isothiocyanates isolated from plants of the Cruciferae family are selectively cytotoxic to tumor cells. It has been demonstrated previously that diisothiocyanate-derived mercapturic acids are highly cytotoxic to colon cancer cells. In the present study, the application of diisothiocyanate-derived mercapturic acids led to a decrease in the viability of an established glioblastoma cell line, primary patient-derived sphere-cultured stem cell-enriched cell populations (SCs), and cells differentiated from SCs. Consequently, targeting glioblastoma cells by diisothiocyanate-derived mercapturic acids is a promising approach to restrict tumor cell growth and may be a novel therapeutic intervention for the treatment of glioblastoma.