The main purpose of this study was to analyze the cytotoxic activity of an extract obtained from Centaurea fenzlii Reichardt, and the fractions eluted from this extract, in breast cancer cells. After isolation and structural analysis of the fractions were conducted, a meaningful cytotoxic effect was indicated. The goal of the analysis was to reveal the mechanism by which this effect occurs through researching the apoptotic side of these fractions and determining the amount of several proteins that are the products of the genes. Test substances were applied to breast cancer cells and the inhibitory concentration value 50 (IC50) that caused a cytotoxic effect was determined using MTT and ATP assays. The Centaurea fenzlii Reichardt dichloromethane extracts-ethyl acetate fractions (CFDCMEAF) exhibited a stronger growth-inhibitory effect on MCF-7 cells (45.771 μg/mL). The apoptotic effect was studied using double staining and flow cytometry. The death rate in the cells treated with the CFDCM-EAF IC50 dose was approximately 90%: 9.2% living cells, 22.8% necrotic cells, 62.3% late apoptotic cells, and 5.8% early apoptotic cells. Structural analysis of the CFDCM-EAF, which indicated significant cytotoxic effects, was performed using chromatographic methods. Hispidulin was the major component of the CFDCM-EAF by LC-APCI-MS/MS analysis.