<p><i>Helicobacter pylori</i> (<i>H. pylori</i>) infection remains a significant global health challenge, contributing to various gastrointestinal disorders, including peptic ulcers and gastric cancer. The bacterium’s urease enzyme plays a pivotal role in its survival by neutralizing gastric acidity, making urease inhibitors a promising therapeutic strategy. This study explores the green synthesis of silver nanoparticles (AgNPs) using Persian lime (<i>Citrus latifolia</i> T.) fruit extract as a reducing and stabilizing agent, optimized via the Taguchi method to achieve minimal particle size for enhanced bioactivity. Characterization techniques such as Fourier-transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD), and UV-Vis spectroscopy confirmed the formation of spherical AgNPs with an average size of 27.63&#xa0;nm and a zeta potential of -13.8 mV, indicating good stability. The green synthesized AgNPs exhibited potent urease inhibitory activity, with an IC₅₀ value of 15.02 ± 2.36&#xa0;µg/mL, surpassing that of Persian lime (<i>Citrus latifolia</i>) fruit extract (IC₅₀= 33.31 ± 1.43&#xa0;µg/mL). In contrast, chemically synthesized AgNPs counterparts exhibited minimal inhibitory activity against urease. These findings highlight the potential of green synthesized AgNPs as effective urease inhibitors with possible relevance to H. pylori-associated pathogenesis. Since H. pylori urease is structurally homologous to the jack bean urease used in this study, these results warrant further investigation using direct anti-H. pylori assays and in vivo models. The integration of natural extracts in nanotechnology not only reduces environmental impact but also enhances biocompatibility, paving the way for novel antimicrobial therapies. Consistently refer to Sample 7 conditions (pH 5, 3500 µL, 5 mM, 80&#xa0;°C) as the experimentally validated optimum, while noting the Taguchi-predicted conditions where appropriate.</p>