Lung cancer incidence is the highest of all cancers, and its management is still a challenge today. Natural anticancer compounds are being developed, one of which is derived from black soybeans. Black soybean (Glycine soja) contains bioactive compounds that are potential anticancer agents. The objective of this research was to determine the secondary metabolite content of black soybean ethanol extract and its toxicity to A549 lung cancer cells. Identification of the content of the extract was performed by thin layer chromatography (TLC) and phytochemical testing. Cytotoxicity testing was performed using an in vitro MTT-assay. The concentration tested were 6.25, 12.5, 25, 50, 100, 200, 400, and 800 μg/ml. The TLC results show that there were 6 compounds in the black soybean ethanol extract. Phytochemical test identified flavonoids, alkaloids, saponins, tannins, triterpenoids, and glycosides that have potential as anticancer agents. The extract exhibited moderate cytotoxic activity in A549 cells (IC50 = 114.5 μg/ml), and the highest extract concentration (800 μg/ml) gave the highest percentage inhibition (83.8%) and significantly different optical density than the other concentrations (p < 0.05). Black soybean ethanol extract has the potential to be developed as a lung anticancer agent. © 2018 Institute of Physics Publishing. All rights reserved.