Fourteen compounds including vanicoside B (1), vanicoside F (2), vanicoside E (3) and 5,6-dehydrokawain (4), aniba-dimer-A (5), 6,6'-((1α,2α,3β,4β)-2,4-diphenylcyclobutane-1,3-diyl)bis(4-methoxy-2H-pyran-2-one) (6), (+)-ketopinoresinol (7), isorhamnetin (8), 3,7-dihydroxy-5,6-dimethoxy-flavone (9), isalpinin (10), cardamomin (11), pinosylvin (12), 2-desoxy-4-epi-pulchellin (13) and β-sitosterol (14) were isolated from dichloromethane-soluble portion of Polygonum hydropiper. By using Alamar blue assay, compounds 2, 7, 8, 11 and 12 were found to be active against Trypanosoma brucei with IC values in the range of 0.49-7.77 μg/mL. Cardamomin (11) had most significant activity against T. brucei with IC/IC values of 0.49/0.81 μg/mL compared to the positive control DFMO (IC/IC: 3.02/8.05 μg/mL). Furthermore, in antimalarial, antimicrobial, anti-inflammatory, PPAR and cytotoxic assays, some compounds have demonstrated moderate inhibitory potentials.