Context: Enicosanthellum pulchrum (King) Heusden (Annonaceae) is a coniferous tree that is confined to mountain forests. The chemical constituents of this species have been studied previously; however, its biological activity has never been investigated before and is reported here for the first time. Objective: The extracts, fractions and compounds from the roots of E. pulchrum were investigated for their inhibitory effects on platelet-activating factor (PAF) receptor binding to rabbit platelets using 3H-PAF as a ligand. Materials and methods: The PAF receptor binding inhibitory effect using rabbit platelets was determined in vitro by measuring the difference between total amount of bound 3H-PAF in the presence and the absence of excess unlabelled PAF. The compounds were isolated by bioassay-guided fractionation and their structures were elucidated by spectroscopic techniques. Results and discussion: Among the extracts tested, the ethyl acetate extract was the most active with 85.6% inhibition, while hexane and methanol extracts showed 40.2 and 42.5% inhibition, respectively. Fractionation of the ethyl acetate extract using vacuum liquid chromatography (VLC) yielded six fractions AEA(I-–VI). Chromatography fraction AEA(VI) yielded a new compound, 1-(2′,3′,4′-trimethoxyphenyl)hexan-1-ol, while fraction AEA(III) afforded three compounds, namely liriodenine, cleistopholine and dehydroanonaine. 1-(2′,3′,4′-Trimethoxyphenyl)hexan-1-ol, cleistopholine and dehydroanonaine showed relatively strong inhibition with IC50 values of 26.6, 50.2 and 45.4 µM, respectively. Conclusion: The results suggest that these compounds could be responsible for the PAF antagonistic activity of the ethyl acetate extract of this plant.