Background: Antibiotic resistance is a worldwide problem that poses a serious threat to human health, limiting the therapeutic options for bacterial infections. The spread of falciparum-resistant malaria is also concerning, making the patient treatment an extremely difficult task. Those facts have heightened the interest to find alternate options to treat infections caused by drug-resistant microorganisms. Objective: Considering the importance of the development of new substances with antibacterial and antimalarial properties, the present study aimed to investigate the activity of the aqueous extract of stem bark of Bowdichia virgilioides (AEBv). This plant is commonly used in Brazilian folk medicine to treat a wide range of illnesses, including signs and symptoms associated with malaria. Materials and Methods: The AEBv was assayed for toxicity against two cell lines and Artemia salina larvae. In vitro activity of the extract was screened against a panel of Gram-positive and Gram-negative bacteria, a chloroquine-resistant (W2) and a chloroquine-sensitive (3D7) Plasmodium falciparum strains. The extract was also tested as antimalarial in vivo against Plasmodium berghei. Results: The AEBv presented no significant toxicity and was found to exert in vitro growth inhibitory effect against the tested bacterial species. The lowest minimal inhibitory concentration was reported for Staphylococcus aureus (0.125 mg/ml) followed by Staphylococcus epidermidis and Staphylococcus saprophyticus (0.50 mg/ml). B. virgilioides extract showed weak in vitro antimalarial activity against P. falciparum. A preliminary phytochemical analysis revealed the presence of flavonoids, phenolic groups, terpenoids, saponins, and tannins and the absence of alkaloids. Conclusion: The AEBv showed promising activity against Gram-positive microorganisms.