A contributing factor in recurrent Listeria monocytogenes (L. monocytogenes) food contamination is that this bacterium produces biofilms on surfaces to persist in food-processing environments. Quorum sensing (QS) is a cell-to-cell communication system utilized by bacteria within biofilms to collaborate and adapt to environmental stresses. However, the details of how the QS-dependent network contributes to biofilm development of L. monocytogenes have yet to be well understood. By comparing the transfer rates of planktonic and biofilm (sessile) L. monocytogenes from stainless steel blades to bologna slices, we found that sessile bacteria had reduced transferability onto a single slice but caused the increase in the number of contaminated slices. This suggests that physiological adaptions derived during biofilm development affect bacterial dissemination. Given the contribution of proteins and environmental temperatures to the extracellular polymeric substances (EPS) synthesis and biofilm integrity, we evaluated the exoproteomes of biofilms formed at 25 and 37°C using 2D-gel electrophoresis and LC-MS/MS. We found exoproteases Lmo0186, Cwh, and Spl exclusively in biofilms formed at 25°C and their greater expression in the gene level at 25°C. By using the zymography and crystal-violet-staining assay with a protease inhibitor, we observed a greater proteolytic activity at lower temperatures and showed that the attenuated proteolytic activity of proteases is positively correlated with increased biofilmorming ability at 25°C. Considering the transcriptional role of QS systems during biofilm development, we investigated how the accessory gene regulator (Agr)-based and metabolite S-Adenosylmethionine (SAM)-involved QS systems modulate nutrient availability and EPS synthesis. The results revealed that the SAM signal interacts with the Agr QS at the transcriptional level during biofilm development, whereas SAM and Agr QS regulate distinct EPS synthesis pathways. Additionally, this interaction is dependent on bacterial life modes (planktonic and sessile). Overall, we conclude that L. monocytogenes manipulates the synthesis of EPS with the coregulation of metabolism and QS for biofilm formation and the production of exoproteases for biofilm dispersion. These precise regulations on EPS enable L. monocytogenes to prolong its survival and promote its dissemination in environments.