This work describes a green synthesis, the characterization, and biological evaluation of silver nanoparticles (AgNPs). The AgNPs suspension was synthesized using aqueous leaf extract of Eucalyptus grandis, which presented a characteristic band at 407 nm in the UV-Vis spectrum. The AgNPs presented a spherical shape and size of 9.7 ± 0.3 nm. The nanoparticles were stable over a month, indicating that E. grandis’ extract is suitable for their preparation and stabilization. The X-ray analysis showed that the crystallinity of AgNPs corresponded to the centered face phase of silver. The antibacterial and leishmanicidal activities of AgNPs were evaluated. The AgNPs presented antibacterial activity on the Gram-negative bacteria Escherichia coli at 53.9 µg mL-1. The leishmanicidal activity evaluation against promastigote forms of Leishmania infantum, Leishmania amazonensis, and Leishmania braziliensis showed that the biological response is dependent on the volume of AgNP suspension. It was demonstrated that L. infantum was more sensitive to the nanoparticle’s treatment than L. amazonensis and L. braziliensis. The treatment of L infantum promastigotes with 150 µL of AgNP suspension reduced parasite growth by 67.9%, a result which was similar to the treatment with 1 (66.7%) or 2 µL (70.6%) of amphotericin B used as a positive control.