Trypanosomiasis is an important but neglected tropical diseases which continues to cause morbidity and mortality on a large scale in sub-Saharan Africa. In the present study, the aqueous leaf extract, flavonoid fraction, and subfractions of the flavonoid from A. senegalensis leaves were evaluated for their antitrypanosomal activities, effects on some hematological parameters, and some liver-based enzymes in mice infected with Trypanosoma brucei brucei. Mice treated with aqueous extract at a concentration range of 100–400 mg kg bw exhibited parasite inhibition range between 66.08 and 73.09% while mice treated with the flavonoid fraction (50-200 mg/kg bw) exhibited higher parasite inhibition (83.37-86.38%). Mice treated with subfractions of the flavonoids showed a significant decrease in parasite count ( p < 0.05 ), with subfraction 3 completely clearing parasites in the circulation after twenty-one days of treatment. Biochemical analysis revealed that the subfraction 3 restored the alterations in the serum aspartate transaminase (AST), alanine transaminase (ALT), and total protein concentrations that were observed in the infected nontreated mice. The subfraction 3 was subsequently characterized by HPLC analysis which revealed the presence of rutin (RT: 22.26, 65.34 ppm) as the most abundant bioactive compound that could be attributed to the bioactivity of the subfraction 3. Other polyphenolic compounds identified included gallic acid (RT: 4.39, 0.578 ppm), quercetin (RT: 37.27, 0.729 ppm), kaempferol (RT: 38.33, 1.52 ppm), and luteolin (RT; 39.173, 32.77 ppm). Taken together, subfraction 3 of the flavonoid fraction from of A. senegalensis demonstrated potent antitrypanosomal activities and therefore represents a reserve of bioactive metabolite worthy of further exploration for the treatment of trypanosomiasis.