The cytotoxicity-guided study of the dichloromethane and ethanol extracts of Thymus mastichina L. using the HCT colon cancer cell line allowed the identification of nine compounds, sakuranetin (1), sterubin (2), oleanolic acid (3), ursolic acid (4), lutein (5), beta-sitosterol (6), rosmarinic acid (7), 6-hydroxyluteolin-7-O-beta-glucopyranoside (8), and 6-hydroxyapigenin-7-O-beta-glucopyranoside (9). All compounds were tested for their cytotoxicity against the HCT colon cancer cell line. Compound 4 showed cytotoxicity with GI50 value of 6.8 microg/mL. A fraction composed of a mixture (1:1) of triterpenoid acids 3 and 4 displayed improved cytotoxicity with a GI50 of 2.8 microg/mL suggesting a synergistic behavior. This is the first report on the chemical constituents of Thymus mastichina L. based on structural assignments by spectroscopic analysis. The presence of these constituents identified by colon cancer cytotoxicity-guided activity indicates that extracts of T. mastichina L. may have a protective effect against colon cancers.