ETHNOPHARMACOLOGICAL RELEVANCE: Amorphophallus konjac (Sheliugu), a medicinal and edible herb from East China and regions south of the Yangtze River, exhibits significant antitumor activity. However, its active constituents and mechanisms remain poorly understood. AIMS OF THE STUDY: This study explores the therapeutic effects of the konjac ethyl acetate fraction (KEAF) in triple-negative breast cancer (TNBC) and elucidates its underlying mechanisms. MATERIALS AND METHODS: UPLC-MS/MS identified KEAF's chemical components, and network pharmacology determined its key targets in TNBC. Survival curves of essential genes were analyzed using UALCAN, bc-GenExMiner, and Kaplan-Meier plotter databases. Protein expression and prognostic data identified TNBC-linked genes. Molecular docking assessed binding affinities of KEAF's bioactive components with these genes. In vitro experiments validated KEAF's effects on proliferation, migration, cell cycle regulation, and apoptosis. RESULTS: KEAF contained 15 active compounds and 146 principal targets, with eight key targets identified: TP53, EGFR, AKT1, MYC, STAT3, HIF1A, ESR1, and JUN. GO and KEGG enrichment analyses highlighted the PI3K/Akt signaling pathway as central to KEAF's therapeutic effects. Protein expression and prognostic studies confirmed EGFR and ESR1 as critical in TNBC progression. Molecular docking revealed strong binding of scutellarein and genistein to EGFR and ESR1 (T score >
5). In vitro, KEAF inhibited MDA-MB-231 cell proliferation and migration, modulated the cell cycle, and induced apoptosis by downregulating PI3K/Akt signaling. CONCLUSION: Scutellarein and genistein in KEAF exhibit therapeutic potential against TNBC by targeting EGFR and ESR1 and suppressing the PI3K/Akt signaling pathway.