Green tea leaf extract-mediated silver nanoparticles (GT-AgNPs) were synthesized and characterized for their biological and chemotherapeutic activity. The synthesis process involved the reduction of silver ions by green tea leaf extract (GTLE), resulting in the formation of GT-AgNPs. Characterization techniques including UV–Vis spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-ray diffractometry (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDX), X-ray photoelectron spectroscopy (XPS), and zeta potential analysis were employed to study the physicochemical properties of GT-AgNPs. The GT-AgNPs exhibited a characteristic color change and a noticeable shift in the surface plasma resonance (SPR) absorption peak at 458 nm, confirming their successful formation of nanoparticles. XRD analysis indicated a cubic structure with a crystallite size of 9 ± 1 nm. Both SEM and TEM images revealed that the spherical shape and size of the synthesized nanoparticles. The SEM analysis confirmed that the average grain size of GT-AgNPs is 20.27 ± 1 nm, while the TEM analysis determined the average particle size of 9 ± 0.025. GT-AgNPs have a negative zeta potential of -19.7 mV indicating a hydrodynamic diameter of 140.90 nm with a polydispersity index (PDI) of 25.8 %. XPS confirmed the oxidation state of Ag0/+1 in GT-AgNPs. The energy band gap (Eg) value of GT-AgNPs is 3.38 eV, indicating semiconducting properties, while fluorescence activity was observed at 506.17 nm. CT-DNA and BSA protein binding studies demonstrated the formation of CT-DNA-GT-AgNPs and BSA-GT-AgNPs complexes with binding constants of 1.366×1010 M−1 and 6.77×109 M−1, respectively. GT-AgNPs exhibited exceptional anti-oxidant activity by effectively scavenging stable 2, 2ʹ - diphenyl-1-picryl hydroxyl (DPPH) radicals. Cytotoxicity assays using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) demonstrate moderate activity against KB3 and AGS cancer cells while showing no detectable cytotoxicity in Caco-2 cells. This indicates a safe therapeutic window for human eukaryotic cells.