Introduction–A previous study showed the unique character of Nigerian red propolis from Rivers State, Nigeria (RSN), withregards to chemical composition and activity againstTrypanosoma bruceiin comparison with other African propolis.Objective–To carry out fractionation and biological testing of Nigerian propolis in order to isolate compounds with anti-trypanosomal activity. To compare the composition of the RSN propolis with the composition of Brazilian red propolis.Methodology–Profiling was carried out using HPLC-UV-ELSD and HPLC-Orbitrap-FTMS on extracts of two samples collected fromRSN with data extraction using MZmine software. Isolation was carried out by normal phase and reversed phase MPLC. Elucida-tion of the compounds with a purity>95% was performed by 1D/2D NMR HRMS and HRLC-MSn.Results–Ten phenolic compounds were isolated or in the case of liquiritigenin partially purified. Data for nine of these correlatedwith literature reports of known compounds i.e. one isoflavanone, calycosin (1); two flavanones, liquiritigenin (2) andpinocembrin (5); an isoflavan, vestitol (3); a pterocarpan, medicarpin (4); two prenylflavanones, 8-prenylnaringenin (7) and6-prenylnaringenin (8); and two geranyl flavonoids, propolin D (9) and macarangin (10). The tenth was elucidated as a previouslyundescribed dihydrobenzofuran (6). The isolated compounds were tested againstTrypanosoma bruceiand displayed moderateto high activity. Some of the compounds tested had similar activity against wild typeT. bruceiand two strains displaying pent-amidine resistance.Conclusion–Nigerian propolis from RSN has some similarities with Brazilian red propolis. The propolis displayed anti-trypanosomal activity at a potentially useful level.