Background: Since there are many researches of phytochemical and bioactivities from genus Alternanthera but did not have any reports from Alternanthera ramosissima. Objective: The objective of the study is to isolate, identify, and determine the bioactivities of the chemicals in this plant, which has not been reported. Materials and Methods: Bioactivity studies of A. ramosissima ethanol extracts were as cytotoxic activity against human cancer cells (human breast carcinoma cell (MCF-7), human cervical carcinoma cell (KB), human cervix adenocarcinoma cell (HeLa) and human colon adenocarcinoma cell (HT-29)) and anti-inflammation. Chromatographic and spectroscopic techniques were used for the isolation and identification of pure compounds. Results: A. ramosissima extracts had the potential effects of cytotoxic activity against human cancer cells and anti-inflammation. Six compounds were isolated; two mixture compounds as the mixture of β-sitosterol and stigmasterol, the mixture of spinasterol and stigmast-7-en-3 β-ol; and four pure compounds as β-sitosterol-D-glucoside, 7-O-β-D-glucopyranosyl chrysoeriol, 7-O-(6”-O-Acetyl)-β-D-glucopyranosyl chrysoeriol, and kaempferol-3-O-β-rutinoside. The isolated compounds were determined the cytotoxic activity against human cancer cells and anti-inflammatory activity. The results showed that all of them did not have cytotoxic activity against human cancer cells. However, the isolated compounds; 7-O-β-D-glucopyranosyl chrysoeriol and 7-O-(6”-O-Acetyl)-β-D-glucopyranosyl chrysoeriol exhibited the potential effect of anti-inflammation by nitric oxide inhibition with IC50as 25.30 and 39.81 μM, respectively, when compared with the positive standards; indomethacin, caffeic acid phenethyl ester, and L-nitroarginine which showed nitric oxide inhibition with IC50as 50.30, 5.62, and 61.80 μM, respectively. Conclusion: This is the first report of chemicals and bioactivities of A. ramosisima and the first report of cytotoxic and anti-inflammatory activities of isolated compounds; 7-O-β-D-glucopyranosyl chrysoeriol and 7-O-(6”-O-Acetyl-)-β-D-glucopyranosyl chrysoeriol.