The food-borne pathogen Listeria monocytogenes can attach to environmental surfaces and form biofilms which can be a source of food contamination, yet little is known about the molecular mechanisms of its biofilm development. The overall objective of this study was to identify the genetic requirements of biofilm formation byL. monocytogenes. In-frame deletion mutants of a putative mutarotase gene (lmo2476/lin2619 ) were constructed to investigate its influence on Listeria biofilm formation. No biofilm phenotype changes were observed between the wild type and the corresponding mutants, indicating that the putative mutarotase gene was not involved in Listeriabiofilm formation under the conditions tested. A mariner -based transposon mutagenesis was performed to generate mutants of L. monocytogenes. A mutant library consisting of 6,500 colonies was screened for reduced biofilm formation. A total of 24 distinct loci were identified, 18 of which, to our knowledge, have not been previously reported to function in the biofilm formation of L. monocytogenes. A putative DNA translocase gene, lmo1386 , was further characterized. The mutant was complemented, and the complemented mutant restored its biofilm phenotype. The lmo1386 mutants showed reduced initial attachment abilities, and had higher numbers of elongated cells when grown in a nutrient TSBYE broth. However, the exact mechanisms of how lmo1386 affects biofilm formation remain to be elucidated. The inhibitory effects of EDTA against biofilm formation of L. monocytogenes were investigated. EDTA at a concentration of 0.1 mM efficiently inhibited biofilm formation of L. monocytogenes without affecting its planktonic growth. EDTA functions in the early stage by affecting the initial attachment of L. monocytogenes cells to surfaces, though the mechanisms remain unclear. The role of extracellular DNA (eDNA) in the formation of L. monocytogenes biofilm was determined indirectly by treatments of DNase I. Our data adds to the knowledge that eDNA plays an essential role in attachment and maintenance of L. monocytogenes biofilm. The pre-formed biofilms on the wells of microtiter plates could be efficiently removed by DNase I, suggesting a potential use of DNase I to eradicate the existing L. monocytogenes biofilms.