Abstract This study has evaluated the possible role played by the l-arginine-nitric oxide pathway in the vasorelaxant action of the hydroalcoholic extract from Eugenia uniflora, and fractions from the extract, in rings of rat thoracic aorta. The addition of an increasing cumulative concentration of hydroalcoholic extract from E. uniflora (1–300 μg mL−1) caused a concentration-dependent relaxation response in intact endothelium-thoracic aorta rings pre-contracted with noradrenaline (30–100 nm). The IC50 value, with its respective confidence limit, and the maximum relaxation (Rmax) were 7.02 (4.77–10.00) μg mL−1 and 83.94 ± 3.04%, respectively. The removal of the endothelium completely abolished these responses. The nitric oxide synthase inhibitors Nω-nitro-l-arginine (l-NOARG, 30 μm) and Nω-nitro-l-arginine methyl ester (l-NAME, 30 μm), inhibited the relaxation (Rmax) to −10.43 ± 7.81% and −3.69 ± 2.62%, respectively. In addition, l-arginine (1 mm), but not d-arginine (1 mm), completely reversed inhibition by l-NOARG. Methylene blue (30 μm), a soluble guanylate cyclase inhibitor, reduced the relaxation induced by the extract to 14.60 ± 7.40%. These data indicate that in the rat thoracic aorta the hydroalcoholic extract, and its fractions, from the leaves of E. uniflora have graded and endothelium-dependent vasorelaxant effects.