This study assessed the effectiveness of methylene blue (MB) and malachite green (MG) on photodynamic inactivation (PDI) of Escherichia coli. The photosensitizers methylene blue (1000 mol L-1) and malachite green (250 mol L-1) were activated with a red light-emitting diode (LED) lamp (max = 636 nm). Bacterial suspensions containing 106 CFU mL-1 were irradiated for 5, 10 and 15 minutes (energy density = 119.9 J cm-2, 223.9 J cm-2 and 335.8 J cm-2, respectively). The following experimental conditions were performed for each photosensitizer: no light irradiation or photosensitizer, irradiation only, photosensitizer only or irradiation in the presence of a photosensitizer. Next, serial dilutions were prepared and seeded onto PCA medium for the determination of the number of colony-forming units per milliliter (CFU mL-1). The results were subjected to analysis of variance (ANOVA) and Tukey test (P<0.05). Photodynamic inactivation using MB and MG was effective in reducing the number of E. coli. Malachite green (250 µmol L-1) photosensitization was able to achieve reductions of over 89% in the viable counts after 15 min of irradiation and methylene blue (1000 µmol L-1), at the same conditions of irradiation, showed a rate growth inhibition of 94.6%. The red LED light used has proven to be effective in the photosensitizing dyes and proved a good alternative to conventional light sources such as laser.