Background: Matricaria chamomilla is an aromatic plant with antioxidant, anticancer, and anti‑inflammatory properties. However, the inhibitory role of M. chamomilla on migration and invasion of human breast cancer cells remains unclear. gThis study investigated the methods to evaluate these anticancer mechanisms of M. chamomilla on human breast cancer MCF‑7 and MDA‑MB‑468 cell lines. Materials and Methods: The cells were treated with hydroalcoholic extract of M. chamomilla at different concentrations (50–1300 μg/mL) for 24, 48, and 72 h in a culture medium containing 10% fetal bovine serum. This study quantified the 50% growth inhibition concentrations (IC50) by 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay; apoptosis and necrosis through Hoechst 33342/propidium iodide staining; cell proliferation and clone formation by clonogenic assay as well as cellular migration, invasion, and attachment. After 24, 48, and 72 h of treatment, the IC50 levels were 992 ± 2.3 μg/mL, 893 ± 5.4 μg/ mL, and 785 ± 4.8 μg/mL against MDA‑MB‑468, respectively, and 1288 ± 5.6 μg/mL, 926 ± 2.5 μg/mL, and 921 ± 3.5 μg/mL, against MCF‑7, respectively. Furthermore, increasing the extract concentrations induced cellular apoptosis and necrosis and decreased cellular invasion or migration through 8 μm pores, colonization and attachment in a dose‑dependent manner. Results: It indicated time‑ and dose‑dependent anti‑invasive and antimigrative or proliferative and antitoxic effects of hydroalcoholic extract of aerial parts of chamomile on breast cancer cells. Conclusion: This study demonstrated an effective plant in preventing or treating breast cancer.