Due to the emergence of multidrug-resistant bacteria, silver nanoparticles (AgNPs) have found interest as a new category of antibacterial agents. The toxicity of the chemicals involved in the commonly employed chemical methods for synthesizing AgNPs present limitations for subsequent pharmaceutical and biomedical applications. In this report, 70% aqueous ethanol extracts of Polygala tenuifolia root were used to reduce Ag+1 ions for AgNPs synthesis. The as-synthesized AgNPs were characterized via UV-Visible spectrophotometry, high resolution transmission electron microscopy, atomic force microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. A strong surface plasmon resonance band was observed at 414 nm. Images from the high resolution transmission electron microscopy and atomic force microscopy demonstrated the spherical and irregular shapes of the AgNPs were synthesized. The AgNP crystalline structure was confirmed by the strong diffraction peaks in the X-ray diffraction results and by the bright circular spots observed in selected-area electron diffraction, whose average diameter was measured to be 17.97 8.63 nm or 15.12 nm via high resolution transmission electron microscopy images or X-ray diffraction analysis, respectively. The as-synthesized AgNPs exerted the highest antibacterial activity against Escherichia coli among the tested Gram-positive and Gram-negative bacteria. The current method is eco-friendly, straightforward, cost-effective, biocompatible, and easily scaled up to produce of AgNPs for applications in the treatment of bacterial infections.