In the present study, silver nanoparticles (AgNPs) were obtained by a green synthesis method by using the aqueous extract of Ilex paraguariensis in two different concentrations. AgNPs were characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM), dynamic light scattering (DLS), Zeta potential, UV-Vis, and FT-IR spectroscopy, and evaluated regarding their potential antimicrobial effects against Escherichia coil and Staphylococcus aureus. AgNPs dry diameter (in height as measured by AFM) was smaller for the sample produced with the lowest extract concentration, varying in number from 4 to 30 nm, while for the sample synthesized with the highest extract concentration, it was between 24 and 60 nm. TEM analyses showed AgNPs with triangular, hexagonal and spherical morphologies, as opposed to AFM results in that the particles were all spherical. Both tested extract concentrations provided a relatively low polydispersity index (0.2-0.3). Moreover, samples presented reproducibility and sustained colloidal stability over the studied time (one year) and a negative Zeta potential. The presence of extract compounds as well as silver nitrate in synthesized AgNPs was indicated by FT-IR assignments. In addition, both AgNPs similarly inhibited the growth of Gram-negative and Gram-positive bacteria at 256 mu M. In sum, the phytosynthesis method using aqueous extract of I. paraguariensis acted simultaneously as a reducing and stabilizing agent to the effective formation of AgNPs with different sizes and shapes; and the particles obtained can be advantageous in applications as antibacterial agents.