Abstract Helicobacter pylori is a bacterium that reaches half of the world population and it's recognized as the main cause of chronic gastritis and peptic ulcer. In this study, we evaluated the anti-H. pylori, antioxidant and immunomodulatory activities of the methanolic (MeOH) extract of Eugenia uniflora leaves and chemical profile. Anti-H. pylori activity was evaluated by spectrophotometric broth microdilution technique by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), in addition to the evaluation of the effect on the urease enzyme. The antioxidant activity was evaluated by capturing O2 •-, HOCl e NO• radicals. The immunomodulatory effect was evaluated on the cytokines TNF-α, IL-6 and on nitric oxide through inhibition in LPS-stimulated macrophages. The chemical profile was performed by total phenolic, tannin and flavonoid contents and mass spectrometry analysis by ESI-FT-ICR MS. In the anti-H. pylori assay the extract showed MIC of 128 μg/mL, however it did not obtain MBC. The extract also showed ability to inhibit the urease enzyme about 20%. The antioxidant activity of the MeOH extract showed EC50 values of 29.77 µg/mL, 15.71 µg/mL and 442.10 µg/mL to O2 •-, HOCl and NO•, respectively. The extract also showed influence on the release of TNF-α, IL-6 and NO in LPS-stimulated macrophages, ranging from 39% to 97% inhibition. Flavonoids, phenylpropanoids, tannins, triterpenoids and carbohydrates were the major classes of compounds present in the MeOH extract as identified by (-)-ESI-FT-ICR MS. The results indicate important anti-H. pylori, antioxidant and immunomodulatory activities from Eugenia uniflora highlighting its importance in the prevention and treatment of diseases caused by H. pylori infection.