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This present study evaluated and rationalized the medicinal use of the fruit part of <i>Acacia nilotica</i> methanolic extract. The phytochemicals were detected using gas chromatography–mass spectrometry (GC–MS) while the in vivo antidiarrheal test was done using Swiss albino mice. To determine the details of the mechanism(s) involved in the antispasmodic effect, isolated rat ileum was chosen using different ex vivo assays by maintaining a physiological environment. GC–MS results showed that <i>A</i>. <i>nilotica</i> contained pyrogallol as the major polyphenol present (64.04%) in addition to polysaccharides, polyphenol, amino acid, steroids, fatty acid esters, and triterpenoids. In the antidiarrheal experiment, <i>A. nilotica</i> inhibited diarrheal episodes in mice significantly (<i>p</i> < 0.05) by 40% protection of mice at 200 mg/kg, while 80% protection was observed at 400 mg/kg by the orally administered extract. The highest antidiarrheal effect was observed with loperamide (<i>p</i> < 0.01), used as a control drug. In the ex vivo experiments, <i>A. nilotica</i> inhibited completely in increasing concentrations (0.3 to 10 mg/mL) the carbachol (CCh; 1 µM) and high K⁺ (80 mM)-evoked spasms in ileum tissues at equal potencies (<i>p</i> > 0.05), similar to papaverine, a dual inhibitor of the phosphodiesterase enzyme (PDE) and Ca⁺⁺ channels. The dual inhibitory-like effects of <i>A. nilotica</i> on PDE and Ca⁺⁺ were further validated when <i>A. nilotica</i> extract (1 and 3 mg/mL)-pre-incubated ileum tissues potentiated and shifted isoprenaline relaxation curves towards lower doses (leftward), similar to papaverine, thus confirming the PDE inhibitory-like mechanism whereas its CCB-like effect of the extract was confirmed at 3 and 5 mg/mL by non-specific inhibition of CaCl₂-mediated concentration response curves towards the right with suppression of the maximum peaks, similar to verapamil, used as standard CCB. Thus, this study characterized the chemical composition and provides mechanistic support for medicinal use of <i>A. nilotica</i> in diarrheal and hyperactive gut motility disorders.