This study is aimed to screen the biological activities and chemical composition to find evidences for potential medicinal applications of Centrostachys aquatica in the Mekong Delta. Crude methanol extract and subextracts in n-hexane, ethyl acetate, and acetone from Centrostachys aquatica were tested bioactivities. The methanol extract, n-hexane and ethyl acetate subextracts exhibited antimicrobial activity with corresponding MIC values of 200, 100 and 200 µg/mL, respectively. The ethyl acetate subextract was inhibited cytotoxicity against cancer cell line LU-1 with IC₅₀ of 27.66 µg/mL. None of the  extracts showed antioxidant ability. Three known secondary metabolites including oleanolic acid (1), 20(E)-hydroxy-b-ecdysone (2), and b-spinasterol (3) were isolated for the first time from the bioactive (ethyl acetate) subextract of Centrostachys aquatica. Their structures were elucidated by modern spectra as MS, NMR and comparison with published data.