Propolis or bee glue, is collected by bees and contains secondary metabolites largely derived from trees or shrubs; it has been used traditionally as a natural remedy with a wide range of biological activities. Its chemical composition is highly complex and variable, and it has been studied in detail worldwide except in Africa. This study investigated the chemical composition and activity of African propolis against blood stream form of Trypanosoma brucei, the causative agent for sleeping sickness that threatens a large population of both humans and animals in sub-Saharan Africa.Extracts of propolis samples (n=12) collected from different regions in Nigeria and one sample collected from South Africa were chemically profiled by using various analytical techniques. These included high performance liquid chromatography (HPLC), coupled with different detection systems including evaporative light scattering detection (ELSD), ultraviolet detection (UV), and high resolution mass spectrometry (HRMS), along with gas chromatography- mass spectrometry (GC-MS) and proton-nuclear magnetic resonance (1H-NMR).Principal components analysis (PCA) of the processed LC-MS data collected was used in order to characterize samples according to their chemical composition. PCA demonstrated the uniqueness in chemical composition of some samples that were also active against Trypanosoma brucei. Therefore, the study proceeded to investigate in detail four samples collected mainly from the southern part of Nigeria. An optimized medium pressure chromatographic technique was used to isolate some of the component(s) responsible for the anti-trypanosomal activity. Two samples collected from Rivers State Nigeria had a different appearance from the rest of the propolis samples, being red in colour and had the highest trypanocidal activity (EC50=4.2 and 6.9 μg/mL) respectively. Their chemical composition was comparable to that of Brazilian red propolis.Fractionation work led to the isolation of ten phenolic compounds including calycosin, liquiritigenen, pinocembrin, vestitol, medicarpin, 8-prenylnaringenin, 6-prenylnaringenin, propolin D, macarangin and a new benzofuran. All compounds structurally elucidated by 1D and 2D Nuclear Magnetic Resonance (NMR) spectroscopy and LC-MSn. Some compounds showed strong inhibitory activity against trypanosomes such as medicarpin (MIC=11.5 μM) and propolin D (MIC=7.4 μM), macarangin (EC50 =18.5 μM), 8-prenylnaringenin (EC50= 17.9 μM), and vestitol (EC50= 30.5 μM). The new benzofuran was moderately active with (EC50=58.01 μM).Fractionation of the propolis sample collected from the Ugelli/Delta sample led to isolation of three compounds 1,3,7-trihydroxy-2,8-di-(3-methylbut-2-enyl) xanthone, 1,3,7-trihydroxy-4,8-di-(3-methylbut-2-enyl) xanthone and a new xanthone. These compounds were tested against T. brucei and presented high activities of EC50= 3.9, 11.04, 14.7 μM respectively.Triterpenes were the main fingerprint compounds in a sample collected from Ijebu-Ode/Ogun; three compounds were isolated and elucidated as ambonic acid, mangiferonic acid and α-amyrin. These compounds had EC50 values against T. brucei of 39.5, 25.5 and 20.9 μM respectively.Finally, sample D46SA from South Africa was found to contain mainly flavonols and diterpenic acids; three compounds pinocembrin, acetylimbricatolic acid and (-)-pimara-8 (14), 15-dien-19-oic acid were isolated and tested. All were moderately active against T. brucei.with MIC ranging from 41.4-137.3 μMIn conclusion, this work has proved the variabiliy of propolis collected even from the same region and the widespread activity of propolis against (blood stream form) T. brucei. It is likely that some of the propolis samples contain compounds with even higher activity that have not yet been isolated.