Background: Breast cancer (BC) is the most common cancer in women, and its prevalence has increased dramatically in recent years. Many treatments for BC have been proven, one of which is the utilization of natural products and herbal derivatives. Among natural plants, plants of the Apiaceae family like Eryngium have been studied, which entail antioxidant, antimicrobial, and most importantly, anti-cancer properties. Objectives: Considering the cytotoxical effects of different Eryngium species, it seems reasonable to evaluate the cytotoxic activity of E. thyrsoideum fractions on the BC, including MCF-7 and MDA-MB-231 cell lines. Methods: The shoots of E. thyrsoideum were extracted by Soxhlet apparatus with n-Hexane, methanol, and dichloromethane solvents. Methanolic extract (strong extract) on C18 Sep-Pak column with a mobile phase of methanol-water was fractionated. Then, the cytotoxicity of different fractions of the strong extract against normal and BC cells was evaluated for 24 and 48 hours using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Induction of apoptosis was assessed by flow cytometry using staining of cells treated with Annexin V/PI. Results: The 80% fraction of the methanolic extract illustrated the strongest cytotoxic effectiveness among the others. This strong fraction specifically prohibited the MCF-7 and MDA-MB-231 growth with minimal effect on normal cells. The prohibition of cell growth had a time- and dose-dependent manner (P < 0.001). In addition, the flow cytometric analysis indicated that the strong fraction exerted its cytotoxic effects by inducing apoptosis on the cancer cell line. Conclusions: According to our results, due to effective secondary metabolites, 80% fraction of methanolic extract prohibited the growth of both types of BC cells by inducing apoptosis and had less toxicity on normal cells.