Epithelial ovarian cancer (EOC) is one of the most malignant gynecological tumors with a high mortality rate owing to tumor relapse after anticancer therapies. It is widely accepted that a rare tumor cell population, known as cancer stem cells (CSC), is responsible for tumor progression and relapse; intriguingly, these cells are able to survive nutrient starvation (such as in vitro culture in the absence of glucose) and chemotherapy treatment. Recent data also indicated that chemotherapy resistance is associated with autophagy activation. We thus decided to investigate both in vitro and in vivo the autophagic activity and the effects of the perturbation of this pathway in CSC isolated from EOC ascitic effusions. Ovarian CSC, identified according to their CD44/CD117 co-expression, presented a higher basal autophagy compared with the non-stem counterpart. Inhibition of this pathway, by in vitro chloroquine treatment or CRISPR/Cas9 ATG5 knockout, impaired canonical CSC properties, such as viability, the ability to form spheroidal structures in vitro, and in vivo tumorigenic potential. In addition, autophagy inhibition showed a synergistic effect with carboplatin administration on both in vitro CSC properties and in vivo tumorigenic activity. On the whole, these results indicate that the autophagy process has a key role in CSC maintenance; inhibition of this pathway in combination with other chemotherapeutic approaches could represent a novel effective strategy to overcome drug resistance and tumor recurrence.