Allicin is a major component in garlic extract and it gives garlic its characteristic taste and odor. In this study, allicin was extracted from the garlic and used in the synthesis of silver nanoparticles (AgNPs). Allicin exhibited a broader ultraviolet (UV) peak at 240 nm, while high-performance liquid chromatography (HPLC) yielded one prominent peak corresponding to allicin. The allicin-mediated AgNPs (Al-AgNPs), and chemically synthesized AgNPs (C-AgNPs) were characterized by ultraviolet–visible (UV-Vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), particle size analyzer (PSA), zeta potential, and transmission electron microscopy (TEM) analyses. The Al-AgNPs demonstrated good 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity (in vitro), and antioxidant potential in albino mice (in vivo). The reduced glutathione (GSH) and catalase (CAT) activity were significantly elevated (p < 0.05) and the activity of superoxide dismutase (SOD) was depleted (p < 0.05) in some groups. The histopathological analysis and all other findings revealed the safer biological nature of Al-AgNPs in comparison to the C-AgNPs. It is concluded that Al-AgNPs are low toxic and safe for oxidative stress-related biomedical applications.