<p>Gastric cancer is the fourth most common cancer globally and the leading cause of cancer-related deaths in Peru. Current synthetic treatments often fail to distinguish cancerous cells from healthy cells, resulting in severe side effects and drug resistance. This study aimed to evaluate the effects of the chloroform fraction of <i>Dracontium spruceanum</i> bulb (DSBCl) on cancer stem cells (CSCs) from AGS and KATO III gastric cancer cell lines, which represent primary and metastatic cancers. The methanolic extract was prepared and characterized via thin-layer chromatography to isolate the chloroform fraction. CSCs were identified via the CD44 marker and isolated via magnetic assisted cell sorting. The cytotoxic effect of DSBCl was evaluated to determine the IC₅₀, revealing its ability to modulate CSC markers and genes associated with tumorigenesis, chemoresistance, and metastasis. In AGS CSCs, DSBCl reduced CD24 expression and downregulated the expression of genes, including <i>ID1, BCL2L2, ABCC2, NANOG,</i> and <i>OCT4</i>, while it upregulated <i>KLF17, BAX,</i> and <i>KLF4</i>. In KATO III CSCs, DSBCl increased <i>ID1</i> and <i>MYC</i> but decreased <i>BCL2L1</i> and <i>BAX</i>. These findings suggest that DSBCl modulates critical markers and genes in gastric CSCs, highlighting its potential for gastric cancer treatment.</p>