Background: Cancer remains to be a major health problem despite advances in treatment. Chemo- and radiotherapy resistance accounting for cancer recurrence have been recently attributed to a subpopulation of cells within the tumor, namely cancer stem cells (CSCs). Aim: Hence, it is essential to adopt new therapeutic approaches that target these cells. Methods and Results: The black seed extract Thymoquinone (TQ) has shown promising anti-cancer effects on various cancer types. Here, we provide an overview of TQ’s potential in targeting CSCs with emphasis on its mechanism of action and shed light on its development as a future drug for cancer therapy. Conclusion: TQ showed potency against CSCs either alone or in combination with chemotherapeutic agents.