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Several foodborne illness outbreaks caused by Salmonella enterica have been associated with the presence of biofilms, particularly on food contact surfaces. The objectives of the current study were: i) to assess the minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) of Aleppo pine essential oil (APEO) against 5 S. enterica serotypes and their mixture in Tryptone Soy broth at 37 °C and, ii) investigate the antibiofilm activity of two mixtures of 3 hydrolytic enzymes (5% protease, 2.5% α-amylase, and 0.5% lipase or 10% protease, 5% α-amylase, and 1% lipase at 50 °C for 30 min, and APEO at 1, 2 and 4 MIC at 25 °C for 10, 20 or 30 min) as well as sequential use of enzyme mixtures and APEO against S. enterica on stainless steel and plastic surfaces. The results showed that the MIC and MBC values of APEO against S. enterica strains and their mixture were 250–500 μg/ml and 1000-4000 μg/ml, respectively. In general, the antibiofilm activity of enzymes and APEO increased as the concentration increased and exposure time was extended. The results showed that a 1.8 log CFU/coupon reduction (p < 0.05) was obtained using 4 MIC APEO for 30 min at 25 °C. The enzyme-based mixture containing 10% protease, 5% amylase and 1% lipase at 50 °C for 30 min reduced the microbial load of S. enterica in biofilms on stainless steel and plastic surfaces by 2.1 log CFU/coupon (p < 0.05). However, a sequential two-step treatment based on a mixture of 10% protease, 5% α-amylase, and 1% lipase for 30 min at 45 °C followed by 4 MIC APEO for 30 min at room temperature reduced the S. enterica cells in biofilms on stainless steel by 4.6 log CFU/coupon (p < 0.05). These results indicated that enzyme-APEO treatments may be applied as a natural alternative to overcome biofilm formation by S. enterica in the food industry. • MIC and MBC of APEO against Salmonella were 250–500 and 1000-4000 μg/ml, respectively. • Washing surfaces with 4MIC APEO for 30 min at 25 °C reduced S. enterica biofilm by 1.8 log. • Use of enzyme mixture for 30 min at 50 °C reduced S. enterica biofilm on surfaces by 2.1 log. • Sequential use of enzyme mixture and 4MIC APEO yielded 4.6 log reduction in biofilm. • Enzyme-APEO treatments may be used to overcome biofilm formed by S. enterica. [ABSTRACT FROM AUTHOR]