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The present study investigated the synthesis and biological applications of green, economical, and multifunctional silver and gold nanoparticles (TSAgNPs and TSAuNPs) using the ethnomedical important medicinal plant Thespesia lampas for biological activities. Relatively higher levels of antioxidant components were measured in T. lampas compared to the wellknown Adhatoda vasica, and Diplocyclos palmatus suggested the potential of T. lampas for the study. Synthesized TSAgNPs and TSAuNPs were characterized through UV–Vis, XRD, SEM-EDS, HR-TEM, SAED, and FTIR techniques. SEM revealed that TSAgNPs and TSAuNPs were predominantly spherical in shape with 19  ±  7.3 and 43  ±  6.3  nm crystal sizes. The sizes of TSAgNPs and TSAuNPs were found to be12  ±  4.8 and 45  ±  2.9  nm, respectively, according to TEM measurements. The FTIR and phytochemical analyses revealed that the polyphenols and proteins present in T. lampas may act as bio-reducing and stabilizing agents for the synthesis. Synthesized NPs exhibited enhanced scavenging properties for ABTS and DPPH radicals. TSAgNPs and TSAuNPs were able to protect DNA nicking up to 13.48% and 15.38%, respectively, from oxidative stress. TSAgNPs possessed efficient antibacterial activities in a concentration-dependent manner against human pathogenic bacteria, such as E. coli, B. subtilis, P. vulgaris, and S. typhi. Furthermore, TSAgNPs and TSAuNPs showed significant cytotoxicity against FaDu HNSCC grown in 2D at 50 and 100  μg  mL−1 . Tumor inhibitory effects on FaDu-derived spheroid were significant for TSAgNPs > TSAuNPs at 100  μg  mL−1 in 3D conditions. Dead cells were highest largely for TSAgNPs (76.65%  ±  1.76%), while TSAuNPs were non-significant, and Saq was ineffectively compared with the control. However, the diameter of the spheroid drastically reduced for TSAgNPs (3.94 folds) followed by TSAuNPs (2.58 folds), Saq (1.94 folds), and cisplatin (1.83 folds) at 100  μg  mL−1 . The findings of the study suggested the bio-competence of TSAgNPs and TSAuNPs as multi-responsive agents for antioxidants, DNA protection, antibacterial, and anti-tumor activities to provide a better comprehension of the role of phytogenic nanoparticles in healthcare systems. [ABSTRACT FROM AUTHOR]