The present study aimed to investigate cytotoxic compounds of Opopanax hispidus (Friv.) Griseb. through in vitro, in silico, and phytochemical analyses. The cytotoxic activity of O. hispidus was evaluated against four different cancer cell lines – lung (A549), breast (MCF-7), liver (HepG2), and cervix (HeLa) – as well as one healthy cell line (Beas-2B), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. n-Hexane and dichloromethane sub-extracts with the IC50 values of 9.77 ± 0.57 and 7.10 ± 0.78 mg/mL on A549, respectively, were fractionated through chromatographic techniques, and the structures of the isolated compounds were elucidated using various spectroscopic methods. A novel compound, 2',6'-dihydroxy-4'-methoxy-3''-(ɤ,ɤ-dimethylallyl)-4''-hydroxy β-hydroxydihydrochalcone (3), along with three known coumarins – umbelliferone 6-carboxylic acid (1), umbelliferone (2), and nodakenetin (4) – were isolated from the active fractions. Compound 3, which possesses a chalcone structure, exhibited notable cytotoxicity against lung cancer cells with an IC50 value of 13.72 ± 0.80 µM.