Numerous studies have already established that autophagy plays a central role in the survival of all cells, including malignant ones. Autophagy is a central cog in the general mechanism that provides the intracellular proteostasis determining cellular physiological and phenotypic characteristics. The accumulated data show that autophagy largely contributes to cancer cell stemness. Thus, autophagy modulation is considered one of the promising pharmacological targets in therapy aimed at cancer stem cell elimination. However, autophagy is a multi-stage intracellular process that involves numerous protein participants. In addition, the process can be activated simultaneously by various signaling modules. Therefore, it is no small feat to select an effective pharmacological drug against autophagy. Whats more, the search for potential chemotherapeutic agents that could eliminate cancer stem cells through pharmacological inhibition of autophagy is still under way. In the present work, we selected a panel of autophagy inhibitors (Autophinib, SBI-0206965, Siramesine, MRT68921, and IITZ-01), some of whom have been recently identified as effective autophagy inhibitors in cancer cells. Using A549 cancer cells, which express the core stem factors Oct4 and Sox2, we evaluated the effect of these drugs on the survival and preservation of the original properties of cancer stem cells. Among the agents selected, only Autophinib demonstrated a significant toxic effect on cancer stem cells. The obtained results demonstrate that autophagy inhibition by Autophinib downregulates the expression of the Sox2 protein in A549 cells, and that this downregulation correlates with a pronounced induction of apoptosis. Moreover, Autophinib-treated A549 cells are unable to form spheroids, which indicates a reduction in stemness. Thus, among the drugs studied, only Autophinib can be considered a potential agent against cancer stem cells.