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In this study, a total of 150 samples including cloacal, carcass swabs, and neck skin excisions were collected from 50 broiler chickens randomly selected in three Italian poultry slaughterhouses. All the samples were examined for the presence of food-borne pathogens and process hygiene indicators. The efficacy of a destructive method and a non-destructive method for recovering of bacteria was also evaluated. Fifteen Listeria monocytogenes isolates were subjected to serotyping and investigated for the presence of 10 virulence-associated genes using multiplex PCR assays. The strains were further subjected to PFGE and investigated for their adhesion abilities. The average levels of the process hygiene indicators (total viable counts of aerobic bacteria, total coliforms, and Escherichia coli) were lower than other surveys carried out in Italy, on the contrary, we highlighted the occurrence of food-borne pathogens as Staphylococcus aureus, Clostridium perfringens, Campylobacter jejuni, and L. monocytogenes. The recovering of L. monocytogenes serotype 1/2a involved in the majority of recent EU food-borne outbreaks of human listeriosis should be considered a public health concern. Practical applications There are a few studies about the occurrence of the main food-borne pathogens and the presumptive process hygiene indicators in Italian poultry slaughterhouses. In our study, we applied a combined microbiological and biomolecular approach to verify their occurrence, to evaluate the practical application of recovery methods and to characterize eventual Listeria monocytogenes isolates. The slaughterhouses included in our study were comparable for slaughter procedures: our results should be useful for Food Business Operators to demonstrate the effectiveness of the preventative measures.