Biofilms lifestyle allows adaptation and protection from antimicrobials and the defense mechanisms of the immune system of host, which is a crucial factor in persistent infections. These threats to global health have stimulated interest in the development of effective antimicrobial drugs and therapies. Photodynamic Inactivation (PDI) uses a photosensitizer and light to produce reactive oxygen species under aerobic conditions, which damage cellular components, inducing the death of microorganisms. In this work, the photodynamic action of 5,10,15,20-tetra (4-N,N,N-trimethylammoniophenyl) porphyrin (TMAP^{4 +}) on the Gram positive bacterium Staphylococcus aureus and the Gram negative Escherichia coli biofilms was quantified through different methods. Firstly, the plate count technique showed that PDI is more effective on S. aureus than E. coli biofilm, and the photodynamic effect increases with the irradiation time. The crystal violet stain did not allow showing significant differences between biofilms with or without treatment of both bacteria, since this dye stains both living and dead cells and also the exopolysaccharides matrix. On the other hand, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method showed that there was a decrease in the number of viable cells as the time of exposure to light increased. Finally, staining with 1,9-dimethyl methylene blue (DMMB) determined that as the irradiation time increased, the amount of matrix present in the biofilm decreased. This effect was present in both strains, being more evident in S. aureus. The increase in knowledge about the target of the PDI on biofilms allows the design of more specific control strategies for their efficient eradication.