The current study optimised the numerous experimental parameters required for the generation and stability of silver nanoparticles (AgNPs) by the green synthesis of AgNP utilizing aqueous Rubus sanctus leaf extract. By employing UV-visible analysis to find the surface plasmon resonance band at 436.5 nm, the creation of nanoparticles was verified. Based on the Fourier transform infrared spectroscopy (FT-IR), the phytochemical components present in the leaf extract of Rubus sanctus significantly impacted the synthesis of AgNPs as a bioreducing agent. The shape, size, and elemental composition of AgNPs were examined using X-ray diffraction (XRD), energy dispersive X-ray (EDX), scanning electron microscope (SEM), and transmission electron microscope (TEM), and the results showed that the particles were crystalline, spherical silver nanoparticles. Additionally, AgNPs' inhibitory effects on the growth of pathogenic Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa bacteria, and Candida albicans yeast were evaluated using the microdilution technique. The results showed that nanoparticles worked even at low concentrations. To test the cytotoxicity of biosynthesised AgNPs, three cancer cell lines, the prostate (LnCap), breast (MDA-MB231), and colon (Caco2), as well as a healthy human embryonic kidney (HEK293) cell line, were used. At high dosages, it was observed that Rubus sanctus-AgNPs had potent cytotoxic activity on the cancer cell lines LnCap, MDA-MB231, and Caco2, but their cytotoxicity was less on the control HEK293 cell line. From the data, it can be inferred that biogenic AgNPs were functionalised, and these compounds demonstrated significant potential as antibacterial and anti-cancer agents for industrial and medical applications.