Aims The purpose of this study was to quantify the extracellular matrix of Listeria monocytogenes biofilm. A preliminary study was carried out to establish a relationship between phylogenetic lineage of 27 strains and their ability to form biofilm in various conditions. Methods and Results Biofilm formation on microtitre plates of 27 strains of L.monocytogenes belonging to lineages I or II was evaluated in different conditions [two temperatures (37 and 22 degrees C) and two media (tryptone soy broth yeast extract medium (TSBYE) and MCDB 202 defined medium)] using crystal violet assay. Lineage II strains produced significantly more biofilm than lineage I strains. In microtitre plates assay, biofilm quantities were greater in MCDB 202 vs TSBYE medium [confirmed by scanning electron microscopy (SEM) analysis] and at 37 vs 22 degrees C. Cultivable bacteria from biofilm population on Petri dishes were enumerated in greater quantities in TSBYE than in MCDB 202 medium. The SEM investigation established that L.monocytogenes biofilms produce extracellular matrix in both media at 37 degrees C. The amount of exopolymers in the extracellular matrix and the pH values were significantly higher in TSBYE than in MCDB 202 medium. The exception was the ScottA strain that presented similar pH values and exopolymer contents in both media. Proteins were the most abundant exopolymer components, followed by DNA and polysaccharides. Conclusions The interpretation of results of biofilm quantification was depending on the growth conditions, the viability of the bacteria and the analysis method. The quantities of proteins, DNA and polysaccharides were different according to the strains and the medium. Significance and Impact of the Study This study screened the potential of a wide panel of L.monocytogenes strains to synthesize exopolymers in biofilm growing condition. The characterization of L.monocytogenes biofilm composition may help to develop new strategies to prevent the formation of biofilms and to remove the biofilms.