The purpose of this research was to develop a system for cultivating and evaluating Listeria monocytogenes biofilms that produces consistent and reliable results. A three-tiered approach was used to evaluate biofilm-forming abilities of three L. monocytogenes strains that were originally associated with listeriosis outbreaks. A L. monocytogenes 'honeycomb' biofilm structure was described. L. monocytogenes strains Scott A and V7 were comparable in developing biofilm network structures and F2365 was less effective in forming biofilm. This three-tiered method can be very useful for further biofilm studies.