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The main objective of this study was to evaluate the efficacy of thymol in controlling environmental contamination in food processing facilities. The effect of thymol was tested as an agent to prevent planktonic and bacterial biofilm growth of twenty-five <i>Listeria</i> <i>monocytogenes</i> isolates from a variety of foods and five <i>Escherichia coli</i> isolates from a farm. The <i>E. coli</i> isolates were positive for extended spectrum &#946;-lactamase (ESBL) genes. All isolates and reference strains were susceptible to thymol at Minimum inhibitory concentration (MIC) values ranging from 250 to 800 &#956;g/mL. An interesting activity of interference with biofilm formation of <i>L. monocytogenes</i> and <i>E. coli</i> was found for thymol at sub-MIC concentrations of 200, 100, 75, and 50 &#956;g/mL. Anti-biofilm activity ranging from 59.71% to 66.90% against pre-formed 24-h-old <i>L.</i> <i>monocytogenes</i> biofilms at concentrations of 500 or 800 &#181;g/mL, corresponding to 2&#215; MIC, was determined against free-living forms of six isolates chosen as the best or moderate biofilm producers among the tested strains. The property of thymol to attack <i>L.</i> <i>monocytogenes</i> biofilm formation was also observed at a concentration of 100 &#181;g/mL, corresponding to 1/4 MIC, by using a stainless-steel model to simulate the surfaces in food industries. This study gives information on the use of thymol in food processing setting.