Biofilms provide an environment that protects microorganisms from external stresses such as nutrient deprivation, antibiotic treatments, and immune defences, thereby creating favorable conditions for bacterial survival and pathogenesis. Here we show that the RNA-binding protein and ribonuclease polynucleotide phosphorylase (PNPase) is a positive regulator of biofilm formation in the human pathogenListeria monocytogenes, a major responsible for food contamination in food-processing environments. The PNPase mutant strain produces less biofilm biomass and exhibits an altered biofilm morphology that is more susceptible to antibiotic treatment. Through biochemical assays and microscopical analysis, we demonstrate that PNPase is a previously unrecognized regulator of the composition of the biofilm extracellular matrix, greatly affecting the levels of proteins, extracellular DNA, and sugars. Noteworthy, we have adapted the use of the fluorescent complex ruthenium red-phenanthroline for the detection of polysaccharides inListeriabiofilms. Transcriptomic analysis of wild-type and PNPase mutant biofilms reveals that PNPase impacts many regulatory pathways associated with biofilm formation, particularly by affecting the expression of genes involved in the metabolism of carbohydrates (e.g.,lmo0096andlmo0783, encoding PTS components), of amino acids (e.g.,lmo1984andlmo2006, encoding biosynthetic enzymes) and in the Agr quorum sensing-like system (lmo0048-49). Moreover, we show that PNPase affects mRNA levels of the master regulator of virulence PrfA and PrfA-regulated genes, and these results could help to explain the reduced bacterial internalization in human cells of the ΔpnpAmutant. Overall, this work demonstrates that PNPase is an important post-transcriptional regulator for virulence and adaptation to the biofilm lifestyle of Gram-positive bacteria and highlights the expanding role of ribonucleases as critical players in pathogenicity.