In the present study, the relationships between serotype, pathogenic profile and <em>in vitro</em> biofilm formation of 106 <em>Listeria monocytogenes</em> strains, having no epidemiological correlation and isolated from different environmental and food sources, were analyzed. The quantitative assessment of the <em>in vitro</em> biofilm formation was carried out by using a microtiter plate assay with spectrophotometric reading (OD620). The isolates were also submitted to serogrouping using the target genes <em>lmo0737</em>, <em>lmo1118, ORF2819, ORF2110, prs</em>, and to the evaluation of the presence of the following virulence genes: <em>prfA, hlyA, rrn, inlA, inlB, iap, plcA, plcB, actA</em> and <em>mpl</em>, by multiplex PCRs. The 62% of the strains showed weak or moderate <em>in vitro</em> ability in biofilm formation, in particular serotypes 1/2b and 4b, frequently associated with sporadic or epidemic listeriosis cases. The 25% of these isolates showed polymorphism for the <em>actA</em> gene, producing a fragment of 268-bp instead of the expected 385-bp. The deletion of nucleotides in this gene seems to be related to enhanced virulence properties among these strains. Strains belonging to serotypes associated with human infections and characterized by pathogenic potential are capable to persist within the processing plants forming biofilm.