<p>Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by enrichment of cancer stem-like cells, high metastatic potential, and poor clinical outcomes. These stem-like populations contribute to drug resistance, tumor recurrence, and therapy failure, highlighting the urgent need for strategies that effectively target stemness. In this study, we evaluated the anticancer effects of ACF-01, a synthetic derivative combining 4′,6,7-trimethoxyisoflavone (TMF) and catechol, in MDA-MB-231-S cells, a CD44⁺/CD24⁻ subpopulation exhibiting enhanced stemness derived from parental MDA-MB-231 cells. Compared with parental cells, MDA-MB-231-S cells displayed elevated aldehyde dehydrogenase activity, increased self-renewal, enhanced drug efflux, and upregulation of stemness-associated markers. Treatment with ACF-01 significantly reduced these stem-like properties and induced apoptosis, as demonstrated by nuclear morphological changes and activation of apoptosis-related proteins, including caspase-3 and PARP cleavage. Notably, co-treatment with doxorubicin produced synergistic effects, further inhibiting stemness and enhancing apoptotic cell death in vitro, as confirmed by combination index analysis. In xenograft models, the combination markedly suppressed tumor growth, decreased stemness marker expression, and showed no evidence of systemic toxicity. Collectively, these results indicate that ACF-01 effectively targets stemness and sensitizes TNBC stem-like cells to doxorubicin, supporting its potential as a promising adjuvant therapy. These findings provide a rationale for further preclinical evaluation and suggest that ACF-01 may improve therapeutic outcomes in TNBC by overcoming stem cell-mediated drug resistance.</p>