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Laetiporus sulphureus, commonly known as the "sulfur polypore" or "chicken of the woods," has a global distribution and primarily grows on deciduous trees as a pathogen or saprotroph. This fungus is rich in bioactive metabolites which exhibit antimicrobial, antioxidant, and possible anticancer properties. However, its effects on hepatocarcinoma cell lines remain poorly investigated. This study evaluates the cytotoxic, antiproliferative, and antimigratory effects of L. sulphureus ethanolic extract on HepG2 and Huh7 hepatocarcinoma cell lines. The results indicate that L. sulphureus ethanolic extract significantly reduces the viability of HepG2 and Huh7 cells in a time- and dose-dependent manner and diminished colony formation. Experiments identified 15 μg/mL as the working concentration for both cell lines, as it approximated the IC50 while ensuring low toxicity. Western blot analysis reveals that L. sulphureus extract increases the expression of key tumor suppressor proteins (p53, p21, and p27), downregulates the anti-apoptotic protein Bcl-2, and upregulates the pro-apoptotic markers (Bax, cleaved caspase-9 and caspase-3), confirming apoptosis induction. Furthermore, the extract impairs Huh7 cell migration, a key factor in cancer progression, as confirmed by a reduction in Twist protein levels and an increase in E-cadherin expression. These results highlight the potential of L. sulphureus as a promising natural adjuvant for hepatocarcinoma therapy while warranting further investigation into its bioactive components, molecular mechanisms, and clinical applications.