Abstract: Vatica diospyroides Symington is the valuable sources of important curative compounds in human cardiovascular and cancer preventions. In order to investigate root extracts of V. diospyroides, for their composition by chromatography and for their cytotoxic activity against breast cancer cell lines in vitro, Thin-layer Chromatography (TLC) was used to separate the chemical constituents and 3-(4,5-dimethylthaizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the 50% inhibition (IC50) concentrations against human breast cancer cell lines MCF-7 and MDA-MB-468. Specific morphological changes of the cancer cells were observed to further elucidate the induction of apoptosis. The chromatography data indicated that different solvents extracted different numbers of components, so that the extracts varied in their chemical compositions. The acetone extract of roots had the highest yield and was highly active whereas the methanol extract was active against the MDA-MB-468 cell line (IC50 = 12.82 and 28.41 μg mL-1, respectively). The other root extracts were active moderately, or inactive, against MCF-7 and MDA-MB-468. Cell shrinking, membrane blebbing, ballooning and chromatin condensation were detected under phase contrast microscopic observations, in the treated cancer cells, as indications of effects that lead to apoptosis. Our results indicate that V. diospyroides root extracts can be highly cytotoxic against breast cancer cell lines. This encourages identifying and purifying an active compound in the root extracts, in a further study.