Bacterial biofilms are a significant problem in the food industry, as they are difficult to eradicate and represent a threat to consumer health. Currently, nanoparticles as an alternative to traditional chemical disinfectants have garnered much attention due to their broad-spectrum antibacterial activity and low toxicity. In this study, silver nanoparticles (AgNPs) were synthesized by a biological method using a Jacaranda mimosifolia flower aqueous extract and by a chemical method, and the factors affecting both syntheses were optimized. The nanoparticles were characterized by Ultraviolet–visible (UV–Vis) spectrophotometry, Fourier-transform infrared spectroscopy (FTIR), Dynamic light scattering (DLS), X-ray diffraction (XRD), and Transmission electron microscopy (TEM) with a spherical and uniform shape. The antibacterial and antibiofilm formation activity was carried out on bacterial species of Pseudomonas aeruginosa and Staphylococcus aureus with the capacity to form biofilm. The minimum inhibitory concentration was 117.5 μg/mL for the chemical and 5.3 μg/mL for the biological nanoparticles. Both types of nanoparticles showed antibiofilm activity in the qualitative Congo red test and in the quantitative microplate test. Antibiofilm activity tests on fresh lettuce showed that biological nanoparticles decreased the population of S. aureus and P. aeruginosa by 0.63 and 2.38 logarithms, respectively, while chemical nanoparticles had little microbial reduction. In conclusion, the biologically synthesized nanoparticles showed greater antibiofilm activity. Therefore, these results suggest their potential application in the formulation of sanitizing products for the food and healthcare industries.