Cancer is a major health issue around the globe. Chemotherapy and radiation therapy are no longer the standard treatments for patients as these remedies lead to many adverse effects. Thus, the development of alternative effective drugs from lead compounds, especially natural products is obligatory. This study aimed is to isolate cytotoxic phytochemical constituent from a plant Calophyllum inophylum, which has been used traditionally since ancient time. The root of C. inophylum was extracted and subjected for phytochemical constituent isolation. A coumarin, Benjaminin (1) was isolated successfully from the chloroform extract and its structural elucidation was performed by spectroscopy analyses of MS, IR and NMR. The anti-proliferative effect of 1 on four cancer cell lines, including leukemia (K562), stomach (SNU-1), liver (Hep-G2) and lung (NCI-H23) cancer were evaluated by using colorimetric MTT assay. Benjaminin (1) exhibited cytotoxic effects towards all the cancer cells and showed the strongest inhibition towards SNU-1 cell proliferation with an IC₅₀value of 70.42 μM. This outcome of this study revealed that 1 is a potential cytotoxic lead compound that could be developed into anti-cancer drug. Thus, further study on the structure-activity relationship of 1 is highly recommended to improve its anti-proliferative effects against cancer cells.