Finding new natural cytotoxic agents against human breast cancer cells is valuable. Essential oils are complex mixtures of volatile compounds with various biological properties. Thymus daenensis Celak (Avishan-e-denaei in Persian) is one of the endemic Thymus species in Iran. Yet cytotoxic properties of Thymus daenensis Celak essential oil (TDEO) against breast cancer cells is unclear. In this study, the cytotoxic activity of TDEO against MDA-MB-231 human breast cancer cell line was evaluated by MTT assay. Results showed that TDEO induces cell death in the cancer cells. The anti-proliferative activity of TDEO was time and dose dependent and more pronounced against MDA-MB-231 cancer cell compared to L929 normal cells. Further analysis is required to understand the type of cell death and underlying cellular and molecular anticancer properties of TDEO.