The utilization of nanotechnology and biotechnology for enhancing the synthesis of plant bioactive chemicals is becoming increasingly common. The hairy root culture technique can be used to increase secondary metabolites such as tropane alkaloids.Agrobacteriumwas used to induce hairy roots from various explants ofHyoscyamus muticus. The effect of nano-silver particles (AgNPs) at concentrations of 0, 25, 50, 100, and 200 mg/L on tropane alkaloids synthesis, particularly hyoscyamine and scopolamine, was studied in transgenic hairy root cultures. Different types of explants obtained from 10-day-old seedlings ofH. muticuswere inoculated with two strains ofAgrobacterium rhizogenes(15,834 and A4). The antimicrobial activity of an ethanolic extract of AgNPs-induced hairy root cultures ofH. muticuswas tested. The frequency of hairy roots was higher in hypocotyl, root, leaf, and stem explants treated withA. rhizogenesstrain A4 compared to those treated with strain 15,834. In transgenic hairy root cultures, AgNPs application at a concentration of 100 mg/L resulted in the highest total tropane alkaloid production, which exhibited broad-spectrum antimicrobial activity. The study demonstrated the potential of nano-silver as an elicitor for promoting the production of target alkaloids inHyoscyamus muticushairy root cultures, which exhibit high biological activity.