Listeria monocytogenes is a food-borne pathogen that can persist in food processing plants by forming biofilms on abiotic surfaces. The benefits that bacteria can gain from living in a biofilm, i.e., protection from environmental factors and tolerance to biocides, have been linked to the biofilm structure. Different L. monocytogenes strains build biofilms with diverse structures, and the underlying mechanisms for that diversity are not yet fully known. This work combines quantitative image analysis, cell counts, nutrient uptake data and mathematical modeling to provide a mechanistic insight into the dynamics of the structure of biofilms formed by L. monocytogenes L1A1 (serotype 1/2a) strain. Confocal laser scanning microscopy (CLSM) and quantitative image analysis were used to characterize the structure of L1A1 biofilms throughout time. L1A1 forms flat, thick structures; damaged or dead cells start appearing early in deep layers of the biofilm and rapidly and massively loss biomass after 4 days. We proposed several reaction-diffusion models to explain the system dynamics. Model candidates describe biomass and nutrients evolution including mechanisms of growth and cell spreading, nutrients diffusion and uptake and biofilm decay. Data fitting was used to estimate unknown model parameters and to choose the most appropriate candidate model. Remarkably, standard reaction-diffusion models could not describe the biofilm dynamics. The selected model reveals that biofilm aging and glucose impaired uptake play a critical role in L1A1 L. monocytogenes biofilm life cycle.