Bioactive compounds in plants have found a wide application in medicines due to their pharmacological or toxicological effects in man and animals. In vitro anti-plasmodial activity of the leaves of Alstonia boonei was studied to support its traditional use as a remedy for malaria. The ethanol extract from the plant was fractionated into ethyl acetate, chloroform and n-hexane fractions. These were subjected to in vitro anti-plasmodial assays using confirmed Plasmodium falciparum infected blood sample from malaria patients. The leaves extracts and fractions were found to be active against Plasmodium falciparum with percentage elimination range of 40.90 to 81.81%, with the ethyl acetate fraction showing the highest activity. The fractions were monitored using chromatographic techniques and this led to the isolation of a β-Sitosterol, a plant derived sterols also known as phytosterols. The structure of the isolated compound was elucidated using spectroscopic methods including FTIR and NMR. β-Sitosterol has been reported to have antimalarial properties giving credence to the traditional use of this plant for the treatment of malaria.