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The leaves of <i>Ranunculus multifidus</i> Forsk. are traditionally used for the treatment of malaria in several African countries. In the present study, 80% methanol (RM-M) and hydrodistilled (RM-H) extracts of fresh leaves from <i>R. multifidus</i> and its major constituent anemonin were tested for their in vivo antimalarial activity against <i>Plasmodium berghei</i> in mice. Anemonin was also tested for its in vitro antimycobacterial activity against <i>Mycobacterium smegmatis</i> and <i>M. abscessus</i> in a microbroth dilution assay, and bacterial growth was analyzed by OD measurement. The isolation of anemonin from RM-H was carried out using preparative thin layer chromatography (PTLC). The chemical structures of anemonin and its hydrolysis product were elucidated using spectroscopic methods (HR–MS; 1D and 2D-NMR). Results of the study revealed that both RM-M and RM-H were active against <i>P. berghei</i> in mice, although the latter demonstrated superior activity (<i>p</i> < 0.001), as compared to the former. At a dose of 35.00 mg/kg/day, RM-H demonstrated a chemosuppression value of 70% in a 4-day suppressive test. In a 4-day suppressive, Rane’s and prophylactic antimalarial tests, anemonin showed median effective doses (ED₅₀s) of 2.17, 2.78 and 2.70 μM, respectively. However, anemonin did not inhibit the growth of <i>M. smegmatis</i> and <i>M. abscessus</i>.