Biofilm initiation and development is a complex process that includes several major stages. This study examined the process of Listeria monocytogenes biofilm initiation and consequent spatial and physiological patterns formed in the biofilm as the result of bacterial response to a changing environment. This is a first attempt at establishing a link between the spatial organization of biofilm-associated bacteria and major stress factors such as surface bioavailability and nutrient content. Developed linear models allow evaluation of some important parameters of the surface population, such as the critical colony size and the intercolony distance in the case of diffusion-limited nutrient access. Variations in bacterial physiological patterns during biofilm initiation as a function of bacterial population density during surface colonization are reported.