The Centaurea species (Asteraceae) is widely used in folk medicine. However, its apoptotic effects have not been adequately determined. Centaurea lycaonica is an endemic species belonging to the Centaurea L. genus. In this study, methanol (CHM) and dichloromethane (CHD) extracts obtained from the aerial part of C. lycaonica were treated to human cervical carcinoma (HeLa) cells to investigate their cytotoxic effects using Sulforhodamine B (SRB) assay and xCELLigence Real-Time Cell analyzing system, in addition to their apoptotic effects by measuring caspases. As a result, the IC50 value of CHD and CHM on HeLa cells was calculated respectively as 39.27 μg/mL and 516.50 μg/mL. Increased activity of caspase-3 and -9, along with a higher Bax/Bcl-2 ratio, was observed in the HeLa cells at 30 and 100 μg/mL concentrations for CHD and 240 μg/mL for CHM. Significant cytotoxic effects through the mitochondrial apoptotic pathway were shown for the HeLa cells. LC-HRMS determined the phytochemical content of extracts. The major compounds, diosmetin, and apigenin, were detected in the CHD extract, and chlorogenic and quinic acid were detected in the CHM extract. Apoptosis is induced primarily by CHD, which contains a high amount of diosmetin and is a promising candidate for anticancer research.