Objectives This work evaluated the antibacterial activity, cytotoxicity and immunomodulatory effect on human peripheral blood mononuclear cells (PBMCs) promoted by aqueous extract from Conocarpus erectus leaves (AELCe). Methods The extract was characterized by thin layer chromatography and ultraperformance liquid chromatography coupled to mass spectrometry (UPLC‐MS). Cytotoxicity of AELCe (6.25–50 μg/ml) was investigated using annexin V and propidium iodide. Cytokine and nitric oxide levels in PBMCs culture supernatants exposed or not to AELCe (12.5 μg/ml) were determined, and antibacterial activity was evaluated by disc diffusion and broth microdilution methods. Key findings AELCe contained 3′,4′‐OH flavonoids, phenylpropanoglycosides, saponins, polymeric proanthocyanidins and hydrolysable tannins. Moreover, 10 other compounds were identified through UPLC‐MS technique. AELCe did not affect lymphocyte viability at 6.25 and 12.5 μg/ml. IL‐2, IL‐10, TNF‐α, IFN‐γ and nitric oxide was produced in higher levels by cells treated with AELCe. Proliferation and activation of CD8+ T lymphocytes were also stimulated. AELCe showed bacteriostatic activity against clinical and antibiotic‐resistant strains of Staphylococcus aureus (MIC between 250 and 1000 μg/ml). Conclusions AELCe showed a moderate bacteriostatic activity and promoted an immunomodulatory status through higher production of Th1 cytokines, nitric oxide release and T CD8+ lymphocytes stimulation.