Previously the nanoparticles were synthesized by chemical methods which were costly and toxic to bio-systems. Plant extracts provides simpler, eco-friendly and cost efficient method for synthesizing nanoparticles. Lemon peel extract (LPE) was used to synthesize silver nanoparticles (AgNPs) which were evaluated for their antimicrobial effects after optimizing the pH of extract and concentration of both extract and synthesized AgNPs. The characterization of synthesized AgNPs was carried out using Ultraviolet-Visible (UV-Vis) Spectrophotometer, Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). Well diffusion method was used to determine the antimicrobial activities of synthesized AgNPs. The presence of phenols and proteins was assumed to reduce the Ag⁺ ion into silver nanoparticles. The characteristic surface plasmon resonance frequency was observed at 405–425 nm for all varying condition of silver nanoparticles synthesis. Furthermore, results revealed that the synthesized AgNPs remains stable upto 75 days. The average particle size was 2–5 nm, calculated with the help of scherrer’s equation by using XRD data. LPE mediated AgNPs (200 µg/mL) showed significant antimicrobial activity, compared to commercially available nanoparticles while LPE (50 mg/ml) showed no effect. LPE mediated AgNPs might get attention of pharmacists in order to design medicines against different diseases including the infections of bacteria.