In recent years, there has been an increased interest in substances that could inhibit or reduce microbial growth in food products. Olive oil industry by-products, due to bioactive compounds with potential antimicrobial properties such as polyphenols, could be used in carcass treatment to enhance hygienic and quality traits. The assessment of the antimicrobial efficacy of bioactive molecules against pathogens should be determined with in vitro and in situ models since it is not possible to evaluate it directly on carcasses at the slaughterhouse. This study aimed to evaluate the effect of an olive mill wastewater polyphenolic extract against Salmonella enteritidis and Listeria monocytogenes, simulating carcass surfaces using bovine dermis samples that were experimentally contaminated with the selected pathogens. The minimum inhibitory concentration and minimum bactericidal concentration were first determined for S. enteritidis and L. monocytogenes. In situ bactericidal activity assessment was performed using 20 cm2 derma samples contaminated with 5 Log CFU/20 cm2 of S. enteritidis and L. monocytogenes in separate trials. Treatment with the polyphenolic extract was not effective for either microorganism. In order to establish the bacteriostatic activity of the polyphenolic extract, suspensions of about 2 Log CFU/20 cm2 of S. enteritidis and L. monocytogenes were used. Polyphenolic extract treatment was not effective against Salmonella, while for Listeria it allowed microbial growth to delay (around 1 Log CFU/cm2 difference at 3, 7, and 14 days between treated and control groups). Further investigations are needed to evaluate the application of polyphenolic compounds on carcass surfaces and their effects on sensory traits.