The aim of this study was to verify the bactericidal effect and the damage of photodynamic inactivation (PDI) using methylene blue (MB) and tungsten-halogen lamp over Listeria monocytogenes via atomic force microscopy, absorption spectrophotometry, agarose gel electrophoresis, real-time PCR and SDS-PAGE. The obtained data indicated that the viability of L. monocytogenes was ca 7-log reduced by illumination with 10 min tungsten-halogen lamp light under the presence of 0.5 μg mL−1 MB, and this bactericidal activity against L. monocytogenes of PDI increased proportionally to the concentration of MB and the duration of irradiation. Moreover, after irradiation with MB and visible light, the leakage of intracellular contents was estimated by spectrophotometer at OD260 and OD280, which correlated with morphological alterations. Furthermore, genomic DNA cleavage and protein degradation were also detected after PDI treatment. Consequently, breakage of the membrane, damage of the genomic DNA and degradation of bacterial proteins may play an important role in the mechanisms involved in PDI-MB bactericidal activity on L. monocytogenes.