Silver nanoparticles show potent anti-bacterial dominancy against bacteria. Therefore, its use as an anti-bacterial agent can be helpful in the treatment of bacterial diseases. In the medical field, the silver element has wide applications. To a small extent, it is harmful to living beings as well if the doses are not taken with proper prescription with necessary precautions. The limited quantities of these nanoparticles could be greatly patientstient in treating the disease. The medicinal products can be synthesized from nanomaterials and may also be used to treat fungal infections. The biosynthesized nanoparticles are of interest to study due to their eco-friendliness. The silver nanoparticles are synthesized from the spider silk of Crossopriza lyoni, a house spider. Therefore, it’s a novel approach for synthesizing silver nanoparticles from the current inventions in the field of nanotechnology. The current synthesized NPs are 20-30 nm in size with a polydispersity index of -20 mV. The spider silk-based nanoparticles enhance the anti-bacterial potential upto multiple times. Therefore, this study successfully elucidates the anti-bacterial potential against gram-negative bacteria such as Salmonella typhi and Klebsiella pneumoniae at the concentration of 0.6 mg/mL.