Several previous studies have reported the beneficial effects of Achillea millefolium, including its anti-cancer properties. The main objectives of this study were to explore the apoptotic and anti-migratory properties of A. millefolium extract against the highly aggressive MDA-MB-231 breast cancer cell line. A. millefolium was subjected to sequential extraction using various solvents, and these fractions were screened for their anti-proliferative effects using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Hoechst staining, reverse transcription-quantitative polymerase chain reaction, and scratch migration assays were employed to monitor the apoptotic and anti-migration activity of the most promising active extract, A. millefolium dichloromethane fraction (AMDF). Moreover, the chemical composition of AMDF was determined using gas chromatography-mass spectrometry (GC-MS). The results of this study demonstrated that AMDF exerted a significant cytotoxic effect on MDA-MB-231 breast cancer cells, accompanied by the induction of apoptosis through the upregulation of pro-apoptotic genes, including tumour protein p53, BCL2 associated X, caspase 3 and caspase 9. AMDF also demonstrated antimigratory properties in MDA-MB-231 cells. Additionally, GC-MS profiling revealed 22 constituents, with M-maleinimido-acetophenone, longifolenaldehyde, azuleno[4,5-b] furan-2,7-dione, and 1,8-cineole identified as major components. In conclusion, this study provided further evidence supporting the potential utility of A. millefolium as a source for novel treatments targeting breast cancer.