Background: Various methods of isolating compounds from plants have been described previously, which include conventional or modern techniques. A preparative-HPLC (prep-HPLC) system has become one of the most convenient methods, with high purity compound as well as consumes less purification time.Objective: This study is intended to purify compounds from E. pulchrum twig extract using prep-HPLC technique and to test all compounds in several biological activities.Methods: Prior to purification using prep-HPLC, the twig extract was injected onto HPLC to develop the method through its chromatograms. The established method from HPLC was used to separate the constituents using prep-HPLC. Purified compounds were elucidated through NMR and MS methods as well as through comparison with previously reported data. Three different biological activities were then conducted on the compounds, including cytotoxicity, DPPH, FRAP, and disc diffusion assays.Results: Cinnamic acid (1) and two aporphine alkaloids (liridine (2) and lysicamine (3)) have been successfully purified and identified. These compounds were first isolated from Enicosanthellum pulchrum using prep-HPLC. Cytotoxic activity revealed that liridine (2) showed strong inhibition against WEHI-3B leukaemic cells of 8.7 �M after 24 h of treatment. In contrast, cinnamic acid (1) and lysicamine (3) exhibited strong inhibitions in antibacterial activity against Staphylococcus aureus, S. epidermidis, Bacillus cereus, Pasteurella multocida and B. subtilis with more than 15 mm of inhibition zone.Conclusion: These phytochemical findings exhibit three isolated compounds from twig extract of E. pulchrum with diverse biological potential to be developed as new agents. � 2019 Bentham Science Publishers.