Breast cancer is one of the most prevalent cancers among women, characterized by the uncontrolled proliferation of breast epithelial cells. Chemotherapy, a common treatment for breast cancer, is associated with significant side effects, including damage to healthy proliferating cells and the development of drug resistance. These limitations highlight the need for novel therapeutic agents. Natural and plant‐based compounds are promising alternatives in this regard. Thymus daenensis Celak. ( T. daenensis ), a medicinal plant from the Lamiaceae family, is rich in bioactive compounds. In this study, the effects of various concentrations of T. daenensis essential oil on MDA‐MB‐231 and MCF‐7 human breast cancer cell lines were evaluated using the MTT assay in both monolayer (2D) and 3D cell cultures over 24 and 48 h. The finding revealed that T. daenensis essential oil caused cell death in a dose‐ and time‐exposure‐dependent manner. The IC ₅₀ values in 3D cultures were higher than those in monolayer cultures, indicating greater resistance of spheroid cells to cell death. The L929 cell line, used as a model for normal cells, showed higher resistance to the cytotoxic effects of the T. daenensis essential oil, suggesting its selective toxicity toward cancer cells. Furthermore, the effects of T. daenensis essential oil on the activity of extracellular matrix metalloproteinases (proMMP‐2 and −9), key players in cancer invasion and metastasis, were assessed via zymography. T. daenensis essential oil significantly reduced proMMP‐2 activity at concentrations of 0.2 and 0.5 mg/mL and proMMP‐9 activity at 0.5 mg/mL in the MDA‐MB‐231 cell line. gas chromatography‐mass spectrometry analysis identified thymol (32.79%) as the most abundant compound in T. daenensis essential oil, followed by carvacrol (8.307%), p‐cymene (7.024%), β‐caryophyllene (7.023%), and caryophyllene oxide (6.39%). These findings suggest that T. daenensis essential oil has potent inhibitory effects on breast cancer cell proliferation and MMP activity, making it a promising candidate for further research.

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