This study was aimed to investigate the anticancer potential of Euphorbia milii (E. milii) using an exquisite combination of phytopharmacological and advanced computational techniques. The chloroform fraction (Em-C) of E. milii methanol extract showed the highest antioxidant activity (IC50: 6.41 ± 0.99 µg/ml) among all studied fractions. Likewise, Em-C also showed significant cytotoxicity (IC50: 11.2 ± 0.8 µg/ml) when compared with that of standard compound 5-fluorouracil (5-FU) (IC50: 4.22 ± 0.6 µg/ml) against hepatocarcinoma cell line (HepG2). However, in a human cervical cancer cell line (HeLa), Em-C demonstrated a non-significant difference in cytotoxicity (22.1 ± 0.8 µg/ml) when compared with that of 5-FU (IC50: 6.87 ± 0.5 µg/ml). Furthermore, Western blot and qRT-PCR analysis revealed that the suppression of HepG2 cells was the consequence of a tremendous decrease in CDK2 and E2F1 protein expression. The GC–MS analysis of Em-C revealed the unique presence of cyclobarbital (CBT) and benzodioxole derivative (BAN) as major constituents. Furthermore, molecular docking of compounds BAN, CBT, and MBT into the binding site of different molecular targets i.e. cyclin dependent kinase 2 (CDK2), thymidylate synthase (TS), caspase 3, BCL2 and topoisomerase II was carried out. Compounds BAN and CBT have demonstrated remarkable binding affinity towards CDK2 and thymidylate synthase, respectively. Molecular dynamic simulation studies have further confirmed the finding of docking analysis, suggesting that CDK2 and TS can act as an attractive molecular target for BAN and CBT, respectively. It can be concluded that these E. milii phytoconstituents (BAN and CBT) may likely be responsible for anti-invasive activity against HepG2 cells.