Background: Malaria, one of the diseases predominant in the African continent, has been reported to be treated with plants and also some in vitro and in vivo tests have supported this. Vernonia amygdalina belonging to the family of Asteraceae is one of the plants widely used in Nigeria and studied for treatment of malaria and some scientific researchers have validated this claim. Objectives: In the present study, we aimed at isolation of possible compounds from the methanolic stem‑bark of V. amygdalina, elucidation and characterization of the isolated compounds, and carry out antimalarial evaluations of the isolated compounds. Materials and Methods: Isolation of compounds was done using column chromatography technique, elucidation and characterization were done based on infrared, Mass, 1H, and 13C nuclear magnetic resonance spectra. The in vitro antimalarial activity was carried out on the ring stage of the malaria parasite cycle of chloroquine‑sensitive (3D7) and chloroquine‑resistant (Dd2) strains. Results: Five novel compounds were isolated; 4α‑Hydroxy‑n-pentadecanoic acid (CC7), 11α‑ Hydroxyurs‑5,12‑dien‑ 28‑oic acid‑3α, 25‑olide (CC10), 1‑Hen eicosanol‑O‑β‑D‑glucopyranoside (CC19), 10‑Geranilanyl-O‑β‑D-xyloside (AC2A), 6β,10β,14β‑Trimethyl heptadecan‑15α‑olyl‑15‑O‑β‑D‑glucopyrano syl‑1,5β‑olide (CC14), and one new compound; Glucuronolactone (CC3). The compounds CC10, CC19, AC2A, CC14, and CC3 recorded schizont inhibition at different percentages. Compounds CC19, AC2A, and CC14 recorded half‑maximal inhibitory concentration (IC50) values of 10.55 μg/ml, 12.56 μg/ml, and 11.68 μg/ml, respectively. Conclusion: The IC50 values obtained are much higher than that of chloroquine, which is 0.02 μg/ml. These compounds showed antimalarial activity at different levels. The presence and effect of these compounds validate the use of this plant for the treatment of malaria in the traditional medicinal practice of Nigeria.