Lipoteichoic acid (LTA) is an important polymer on the surface of Gram-positive organisms, including the foodborne, biofilm-forming pathogen Listeria monocytogenes, and is connected to the cell membrane through a glycolipid backbone. Previous results have shown the importance of LTA in cell viability and biofilm formation. The purpose of this research is to further investigate the influence of LTA in L. monocytogenes biofilm formation and how the bacterium responds to environmental stress caused by antimicrobial exposures, and a combination of low temperature and high osmolarity. We created in frame deletion mutants along the operon lmo-2555-lmo2554-lmo2553, which have been shown to reduce the ability of L. monocytogenes to form biofilm. We were able to show that our mutant strain had less amount of LTA on their cell membrane and formed less biofilm both on a PVC well plate and drip flow reactor. Visualization of biofilm cells using confocal microscopy also showed that our mutant strain had thinner biofilm compared to its positive control and its complement strain. We believe that the reduction in biofilm formation was due to the ability of cells to attach prior to forming biofilms. Our mutant strains also had increased sensitivity toward anionic and cationic antimicrobials. The influence of LTA was more pronounced when cells were exposed to low temperature, where our mutant strain was not able to grow even after a 14-day incubation at 4oC. Our strains also showed different sensitivity to various humectants at lower temperature (4oC and 20oC), where glycerol was the least harsh, while sucrose was the harshest. The absence of LTA did not seem to influence the cells’ tolerance on high osmolarity in the presence of NaCl. However, our mutant strain showed considerable difference in osmotolerance in the presence of sucrose. Further understanding of biofilm formation mechanism will eventually lead to a more intelligent design of L. monocytogenes biofilm removal in the food processing environment.