a Department of Chemistry, College of Sciences, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia

b Department of Biochemistry, University of Veterinary and Animal Sciences, Lahore (Jhang-campus), Jhang 35200, Pakistan

c Renewable Energy and Environmental Technology Center, University of Tabuk, Tabuk, 47913, Saudi Arabia

d School of Chemistry, University of the Punjab, Lahore, Pakistan

e Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan

f Department of Chemistry, The University of Lahore, Lahore, Pakistan

g Department of Chemistry, Faculty of Sciences at Bisha, University of Bisha, P.O. BOX 199, Bisha 61922, Saudi Arabia

h Department of Chemistry, Division of Science and Technology, University of Education, Lahore, Pakistan

i Institute of Chemistry, The Islamia University of Bahawalpur, Bahawalpur, Pakistan

This study investigates the biological potential of Seidlitzia stocksii-mediated Ag/Cu bimetallic nanoparticles (NPs), synthesized using plant extract as a natural reducing and stabilizing agent. The aqueous stem extract of S. stocksii (SSAE) was evaluated for its phytochemical composition, revealing a total phenolic content (TPC) of 47.91 mg GAE/g and a total flavonoid content (TFC) of 40.78 mg QE/g at the highest tested concentration. The biosynthesized Ag/Cu bimetallic nanoparticles were assessed for their antioxidant, antibacterial, and membrane-stabilizing potential. The NPs were further evaluated for their anti-cancerous potential against HepG2 carcinoma cells, while their mutagenic properties were examined using the Ames test. Antioxidant analysis demonstrated a dose-dependent increase in activity, with DPPH radical scavenging reaching 47.32% and reducing power measured at 38.76%. Antibacterial efficacy was tested against Escherichia coli and Bacillus subtilis, showing a concentration-dependent increase in the zone of inhibition (ZOI). Among the tested strains, E. coli showed the highest susceptibility, with a ZOI ranging from 12 to 23 mm. Hemolytic analysis revealed that erythrocyte lysis remained below the ASTM threshold, with 4.76% hemolysis observed at 120 mg/mL. Cytotoxic evaluation via MTT assays on HepG2 cells indicated 86.87% cell viability, supporting NPs ’potential anticancer activity. Genotoxicity assessment using S. typhimurium strains TA98 and TA100 revealed no significant increase in revertant colony count, confirming the non-mutagenic nature of NPs. Collectively, these findings indicate that S. stocksii-mediated Ag/Cu-NPs exhibit multifunctional biological activities, establishing these NPs as safe, biocompatible agents with antioxidant, antibacterial, and anticancer potential. Future mechanistic and in vivo studies are needed to validate these findings and support their practical application.