Lactococcus carnosus CNCM I-4031 is a psychrotrophic lactic acid bacterium used for the biopreservation of seafood. It effectively inhibits the growth of spoilage and pathogenic bacteria, such as Listeria monocytogenes, through an atypical mechanism that relies on direct cell-to-cell contact, without producing conventional antimicrobial compounds like bacteriocins. However, the precise molecular mechanism behind this bacterial interaction remains to be fully understood. In this study, Label-free LC-MS/MS shotgun proteomics and gene expression analysis were used to examine cell envelope protein expression in L. carnosus when cultivated alone and in co-culture with L. monocytogenes. The investigation identified a specific cell wall protein, named LYSO, which has a toxic C-terminal domain and demonstrates peptidoglycan hydrolysis activity against L. monocytogenes. Further analysis using knockout mutants provided additional evidence for the involvement of LYSO in the inhibition activity. These findings suggest the significant role of this bacteriolysin in the contact-dependent mechanism of L. carnosus against L. monocytogenes.