Durio kutejensis has known potential as herbal medicine in Kalimantan. Many things can be explored from D. kutejensis related pharmacological activity in every part of the plant included root bark. This study investigated the phytochemical content with a qualitative test and tested cytotoxic activities of D. kutejensis root bark on MCF-7 cell lines. Cytotoxic activity tested on MCF-7 cells with MTT assay method. The result showed that D. kutejensis root bark contains flavonoid, tannin, terpenoid, phenol, and saponin. IC50 value for ethanol extracts of root bark, n-hexane fractions, ethyl acetate fractions, and doxorubicin on MCF-7 cells are 761.29; 280.5; 207.08; and 0.25 μg/mL, respectively. In conclusion, D. kutejensis root bark has some secondary metabolites but no cytotoxic activity on MCF-7 cells. Further research on other compounds to be investigated and test to other pharmacological activity.