A series of isatin-dehydroepiandrosterone hybrids were synthesised via a convenient condensation procedure, and which were evaluated for their potential anticancer activities. The preliminary assays indicated that some of the newly obtained compounds exhibited good antitumor activities against human hepatocellular liver carcinoma (HepG2), heptoma (Huh-7), melanoma (A875) and 5-fluorouracil-resistant human hepatocellular carcinoma (BEL-7402/5-FU) cell lines compared with 5-fluorouracil (5-FU), which might be considered as promising lead scaffold for further design and synthesis of highly potential anticancer agents.