Diarrhoea is a gastrointestinal tract disorder characterized by frequent stooling. The use of the aqueous extract of Annona senegalensis root and stem bark for the treatment of diarrhoea has been documented. However, limited information exists on the components, mechanism of action and safety. The aim of the study was to carry out biochemical assessment of the antidiarrhoeal fractions from A. senegalensis root and stem barks in castor oil-induced diarrhoeal rats. The objectives of the study were to: (i) evaluate the antidiarrhoeal activity of solvents extract of A. senegalensis stem and root barks; (ii) fractionate the bioactive extracts; (iii) explore possible mechanism(s) of action of the bioactive fraction(s); (iv) evaluate the toxicity of bioactive sub-fractions; and (v) identify chemical compounds in the bioactive sub-fractions.rnAqueous, n-hexane and dichloromethane stem and root bark extracts of A. senegalensis were screened for antidiarrhoeal activity using castor oil-induced diarrhoeal, gastrointestinal tract (GIT) motility inhibition and antienteropooling models. Bioactive aqueous extract was partitioned into hexane, dichloromethane and ethylacetate fractions. Bioactive fractions were fractionated into sub-fractions using column chromatography. The mechanism(s) of action of the bioactive sub-fractions was determined by evaluating their antioxidant properties, intestinal fluid electrolyte concentration, small intestinal Na+- K+ ATPase and cyclooxygenase II activities. Kidney and liver function indices were evaluated after administration of bioactive sub-fractions. Active principles were identified using gas chromatography-mass spectrometry. Data were subjected to analysis of variance and Duncanâ€™s multiple range test at p < 0.05.rnThe findings of this study were that:rni. aqueous stem (AS) and dichloromethane root (DR) extracts at 100 mg/kg body weight (bw) significantly decreased the number of wet feaces while GIT motility inhibition and antienteropooling activity were significantly increased by aqueous root (AR) and dichloromethane stem (DS) extracts;rnii. inhibition of defecation was highest (85.25%) in the ethylacetate fraction of AS (EFAS), while antienteropooling and GIT motility inhibition was highest in the dichloromethane fraction (DFAR) and ethylacetate fraction (EFAR) of AR respectively;rniii. sub-fraction 1 of EFAS (EFAS1) and fraction 2 of DR (DR2) at 25 mg/kg bw exhibited the highest inhibition of defecation;rniv. anti-motility activity was highest in sub-fraction 2 of EFAR (EFAR2) at 25 mg/kg bw while antienteropooling activity was highest in fraction 3 of DS (DS3) and sub-fraction 1 of DFAR (DFAR1);rnv. EFAS1 had the least IC50 for diphenyl-1-picrylhydrazyl, highest hydrogen peroxide scavenging activity, significantly increased Na+-K+ ATPase activity and decreased cyclooxygenase II activity;rnvi. chloride and Na+ concentration were significantly decreased by EFAR2 and DS3 respectively.rnvii. alanine aminotransferase and Na+ significantly increased while urea decreased at 200 and 400mg/kg bw of DFAR1, EFAR2, EFAS1, DS3 and DR2;rnviii. chemical compounds identified were; catechol and ethyl eicosapentanoate in EFAS1; piperidnyloxy, thiazoline and quinazoline in EFAR2; and aldosterone derivatives in DFAR1 and DS3.rnThe study concluded that antidiarrhoeal principles present in DS3, EFAS1, DR2, EFAR2 and DFAR1 exhibited different mechanism of action. Fourteen days administration of these principles was not safe. Identified bioactive principles may be explored for the development of antidiarrhoeal drugs.