Biofilms provide a suitable environment for L. monocytogenes and are the cause of enormous risks in the food industry. SpoVG is a global regulatory factor that plays a vital role in physiological activity of L. monocytogenes. We constructed spoVG mutant strains to investigate the effects of these mutants on L. monocytogenes biofilms. The results show that L. monocytogenes biofilm formation was decreased by 40%. Furthermore, we measured biofilm related phenotypes to study the regulation of SpoVG. The motility capacity of L. monocytogenes was found to decrease after the deletion of spoVG. The cell surface properties changed in the spoVG mutant strains, with an increase in both the cell surface hydrophobicity and the auto-aggregation capacity after spoVG deletion. SpoVG mutant strains were found to be more sensitive to antibiotics, and had a reduced tolerance to inappropriate pH, salt stress and low temperature. The RT-qPCR results showed that SpoVG effectively regulated the expression of genes related to quorum sensing, flagella, virulence and stress factors. These findings suggest that spoVG has potential as a target to decrease biofilm formation and control L. monocytogenes contamination in the food industry.