The presence of virulence genes, phylogenetic relationships, biofilm formation index (BFI), and ultrastructure in S. Minnesota at different temperatures (4, 25, and 36 °C) were analyzed. In addition, the ability of biocidal agents (chlorhexidine1%, sodium hypochlorite 1%, and peracetic acid 0.8%) to inhibit biofilms formed by 20 strains isolated from broiler slaughter plants from two Brazilian companies in 2009, 2010, and 2014 was determined. The presence of specific genes was evaluated by PCR and phylogeny between strains by pulsed-field gel electrophoresis. The BFI was determined using tryptone soy broth with 5% of chicken juice, and its structure was observed by scanning electron microscopy. The presence of specific genes indicated that S. Minnesota has the potential to cause disease in humans, adapting to adverse conditions. Temperatures of 25 and 36 °C favored biofilm formation, although at 4 °C, there was still biomass that could contaminate the final product. Tolerance to all biocides was identified in 12/20 (60%), representing a real risk of adaptation mechanisms development, especially regarding to resistance to sodium hypochlorite. Phylogenetic analysis indicated cross-contamination and spread among companies, which was probably related to biofilms formation. Results show the necessity of attention to this serovar considering its resistance to sodium hypochlorite, including the need for rigorous control, adopting low temperatures to prevent biofilms formation in the poultry industry.