Introduction: The worldwide investigation of plant species for the treatment of various diseases and infections is still valuable. In developing countries, such as Ethiopia, where health care coverage is less, traditional medicine has played a significant role in treating health problems. Moringa stenopetala is among plant species widely used in traditional medicine for treatments of human health cases. Aims: This study was conducted to isolate bioactive compounds from Moringa stenopetala root for medicinal purposes. Methodology: The dried root of Moringa stenopetala was extracted with different solvents: n-hexane, dichloromethane/methanol (1:1) and methanol using maceration. Results: Phytochemical analysis confirmed the presence of alkaloids, saponins, flavonoids, phenols, steroids, glycosides, terpenoids and tannins. Compounds were isolated and the compounds were characterized using results from spectroscopic techniques (IR, 1H-NMR, 13C-NMR and DEPT-135). The results signify that isolated and characterized compounds are (5E, 7Z, 10E)- 4,5,6,7-tetramethylnonadeca-5,7,10-trienoic acid and (4E,6E,8E)-pentadeca-4,6,8-trienoic acid with a structure that nearly look like simple fatty acids. Conclusion: Fatty acids are known to possess antimicrobial activities of different strains and the result of this work supports the indigenous knowledge of the community of using this tree for antimalarial and anti-pain treatments.