Bombyx mori silk is one of the most extensively studied types of silk due to its unique mechanical properties and biocompatibility, which have enabled its Utilization in medical applications Including surgical sutures since the second century. In the present study, a new method for the biosynthesis of silver nanoparticles (AgNPs) was explored by applying Bombyx mori cocoon extract as a sustainable and eco-friendly biological source. Unlike previous studies that primarily utilized plant or microbial extracts, this approach offers a more efficient alternative due to the unique protein and polyphenol content of silk cocoons, which enhances the stability and biological properties of the biosynthesized nanoparticles. The resulting AgNPs exhibited significant antibacterial, antioxidant, anti-inflammatory, and cytotoxic properties, opening new avenues for their therapeutic applications. This study expands the range of biological materials used in AgNP synthesis and provides deeper insight into how different bioactive components influence their functional properties. In this study, AgNPs were biosynthesized by mechanically processing extracted raw silk material with silver nitrate (AgNO₃). The synthesized nanoparticles were characterized by implementing several physicochemical techniques, including UV-visible spectrophotometry, FTIR, and XRD, and their morphology was examined through Transmission Electron Microscopy (TEM). The obtained AgNPs displayed a distinct absorption peak at 420 nm, with a particle size ranging between 5 and 25 nm, and displayed characteristic FTIR and XRD patterns typical of silver nanoparticles. The biosynthesized AgNPs demonstrated significant antimicrobial activity against Staphylococcus aureus (ATCC25923), Staphylococcus haemolyticus (ATCC29968), Escherichia coli (ATCC8739), and Klebsiella pneumoniae (ATCC2146). The antioxidant potential, assessed via the DPPH assay, yielded an IC50 value of 4.94 µg/ml, while the anti-inflammatory effect, evaluated using the membrane stabilization technique, showed an IC50 of 7.14 µg/ml. Additionally, AgNPs exhibited notable cytotoxic properties against Caco-2 and PANC1 cell lines, with IC50 values of 177.24 ± 2.01 µg/ml and 208.15 ± 2.79 µg/ml, respectively. Conversely, their impact on normal HFB-4 cells was minimal, with an IC50 of 582.33 ± 6.37 µg/ml, indicating a favorable safety profile. These observations highlight the multifunctional potential of silk-derived AgNPs, suggesting their applicability in various biomedical fields.