The incidence of colorectal cancer is the third highest of all cancers in Indonesia and worldwide, with a high mortality rate and varying degrees of therapeutic success. Black soybean extract has potential as an anticancer agent because it has some secondary metabolites, such as flavonoids and alkaloids, that have cytotoxic effects on cancer cells. This study aimed to determine the cytotoxic effect of black soybean ethanolic extract on HCT-116 colorectal cancer cell line. Thin-layer chromatography (TLC), phytochemical screening tests and the MTT assay were performed. TLC revealed that there were six components of different compounds in black soybean ethanolic extract. Phytochemical screening tests identified six classes of chemical compounds, namely, alkaloids, tannins, flavonoids, saponins, triterpenoids and glycosides. MTT assay of black soybean ethanolic extract on HCT-116 cells exhibited a half-maximal inhibitory concentration (IC50) value of 97.56 μg/ml with moderate cytotoxic properties compared with cisplatin, used as a positive control, which had IC50 of 55.51 μg/ml with moderate cytotoxic properties. Based on statistics, there was a significant differences in absorbance values between groups of concentrations (p = 0.001). Black soybean ethanolic extract had a cytotoxic effect on HCT-116 cell growth. © 2018 Institute of Physics Publishing. All rights reserved.