Background: Sphaeralcea angustifolia (Malvaceae) is extensively used in Mexican traditional medicine for the treatment of gastrointestinal disorders such as diarrhea and dysentery. Objective: The current study was to validate the traditional use of S. angustifolia for the treatment of diarrhea and dysentery on biological grounds using in vitro antiprotozoal activity and computational experiments. Materials and Methods: The ethanol extract, subsequent fractions, flavonoids, phenolic acids, and a sterol were evaluated on Entamoeba histolytica and Giardia lamblia trophozoites. Moreover, molecular docking studies on tiliroside were performed; it was tested for its affinity against pyruvate:ferredoxin oxidoreductase (PFOR) and fructose‑1,6‑bisphosphate aldolase (G/FBPA), two glycolytic enzymes of anaerobic protozoa. Results: Bioassay‑guided fractionation of extract of the aerial parts of S. angustifolia gives tiliroside and apigenin, caffeic acid, protocatechuic acid, and β‑sitosterol. The in vitro antiprotozoal assay showed that tiliroside was the most potent antiprotozoal compound on both protozoa with 50% inhibitory concentration values of 17.5 μg/mL for E. histolytica and 17.4 μg/mL for G. lamblia. Molecular docking studies using tiliroside showed its probable antiprotozoal mechanism with PFOR and G/FBPA. In both cases, tiliroside showed high affinity and inhibition constant theoretic for PFOR (lowest free binding energy from −9.92 kcal/mol and 53.57 μM, respectively) and G/FBPA (free binding energy from −7.17 kcal/mol and 55.5 μM, respectively), like to metronidazole, revealing its potential binding mode at molecular level. Conclusion: The results suggest that tiliroside seems to be a potential antiprotozoal compound responsible for antiamoebic and antigiardial activities of S. angustifolia. Its in vitro antiprotozoal activities are in good agreement with the traditional medicinal use of S. angustifolia in gastrointestinal disorders such as diarrhea and dysentery.