To study the chemical constituents and explore the anti-Epstein-Barr virus (anti-EBV) bioactive compound from the aerial part of Breynia fruticosa.The compounds were separated by column chromatography such as silica gel,MCI gel CHP-20,ODS,and Sephadex LH-20 and purified by preparative HPLC method.The structures of all the isolated compounds were identified by combination of spectroscopic methods (MS,¹H NMR,¹³C NMR) with the literature data,then all compounds assay through anti-EBV.As a result,chemical investigation on the aerial part of Breynia fruticosa led to the isolation of 24 compounds.Here,including (-)-syringaresinol (1),(+)-epi-syringaresinol (2),(+)-syringaresinol (3),(+)-(7R,8R,7′S)-lyoniresinol (4),(+)-5-methoxy-isolariciresinol (5),epi-lyoniresinol (6),burselignan (7),(-)-isolariciresinol (8),(-)-(7S,7′S,8R,8′R)-3,3′,5,5′-tetramethoxy- 7,7′-ep-oxylignane-4,4′,9,9′-tetraol (9),(7S,7′R,8S,8′S)-3,3′-dimethoxy-7,7′-epoxy-ligna-ne-4,4′,9,9′-tetraol (10),(+)-grandisin (11),1,2-methylene-dioxy-4-methoxy-secopterocarpan (12),2-［4-(3-hydroxypropyl)-2-methoxy- phenoxy］-propane-1,3-diol (13),shepherdine (14),4-O-methylnorbergenin (15),5-O-methylnorb-ergenin (16),maniladiol (17),olean-12-ene-3β,22β-diol (18),taxaxerol (19),daturadiol (20),dihydrobrassicasterol (21),β-sitosterol (22),(E)-6-methoxyl-decylcaffeate (23),(8E,10Z,15E)-7-hydroxyoctadeca-8,10,15-trieno-icacid (24) were reported in this paper.Except 1,3,4,8,22,all the compounds were obtained from this plant for the first time.The results showed that two compounds inhibited more than 50% cleavage and replication of EB virus with concentrations of 30 μmol/L.The IC₅₀ of compounds 12 and 14 were 16.4 μmol/L and 5.7 μmol/L,respectively.