The proliferation of foodborne pathogens and the formation of biofilms poses a major threat to the food industry, particularly in fresh produce. Raw and minimally processed fruits and vegetables are often sold to consumers in ready-to-use or ready-to-eat forms and are at the forefront of this risk. These products lack preservatives and rarely undergo any heat processing prior to consumption, making them more susceptible to contamination. To address this, we synthesized an oil/water emulsion encapsulating eugenol, using exopolysaccharide extracted from Lactobacillus as a natural emulsifier, and investigated its effectiveness against pathogenic cell populations within biofilm matrices. Different strains of Listeria monocytogenes, Salmonella, and E. coli were studied; results reveal substantial variations in biofilm-forming abilities among different E. coli, Salmonella, and L. monocytogenes strains. Specifically, L. monocytogenes V7 and Scott A strains displayed resistance to the emulsion, resulting in a minimal reduction of 2.8 log CFU/cm2 on the stainless-steel surface within the biofilm. Conversely, other strains exhibited reductions ranging from 3 to 4.5 log CFU/cm2. Most strains’ biofilm inhibition was 85% on plastic surfaces, whereas L. monocytogenes V7, Scott A, and Salmonella enterica ATCC 35664 showed less inhibition. The emulsion was applied to fresh produce (tomatoes, blueberries, and lettuce), where the reduction in E. coli and Salmonella biofilms, along with planktonic cells, varied from 0.53 to 5.1 log CFU/cm2, indicating that the emulsion significantly inhibited the growth of the tested pathogens. In conclusion, the results indicate that biofilm inhibition depends on the microbial species due to the observed individual variation in strains. However, combining eugenol and exopolysaccharide as emulsions is a promising natural alternative and sustainable technology for removing pathogenic biofilms. This research has the potential to advance food safety and hygiene standards, with practical applications in preserving fresh produce and disinfecting surfaces. This work was supported by USDA-NIFA- SCRI- 2017-51181-26834 through the National Center of Excellence for Melon at the Vegetable and Fruit Improvement Center of Texas A
