In this study, the antimicrobial activity of silver nanoparticles (AgNPs L₂₁, AgNPs L₂₂) produced using aqueous extracts of the Laser trilobum harvested in 2021 and 2022 was optimized. The experimental design and optimization of antimicrobial activity were performed using the response surface method (RSM). Using RSM, independent variables, such as silver concentration, extraction time, and synthesis temperature, were optimized as a result of the inhibition zone diameter against Staphylococcus aureus, Escherichia coli, Salmonella Typhimurium, and Listeria monocytogenes. Based on the optimization results, the extraction time, silver concentration, and temperature for AgNP L₂₁ and L₂₂ synthesis were determined to be 60 min, 5 mM, and 25 °C, respectively. When the XRD and TEM results of the nanoparticles synthesized under optimal conditions were evaluated, it was determined that the AgNPs were spherical in shape and had an average size of 30 ± 12 nm. Our study revealed that the year of harvest is unimportant for nanoparticles synthesized from L. trilobum, using a cheap and simple method that does not require toxic substances. Owing to the antimicrobial activity of nanoparticles produced under optimal conditions, it is possible to biocontrol and prevent contamination by these bacteria in food science and industry.