Tissue-specific modification of treatment strategy is proposed to increase the antimicrobial activity of light-activated therapy (LAT) for root canal disinfection. Methylene blue (MB) dissolved in different formulations: water, 70% glycerol, 70% poly ethylene glycol (PEG), and a mixture of glycerol:ethanol:water (30:20:50) (MIX), is analyzed for photophysical, photochemical, and photobiological characteristics. Aggregation of MB molecules, as evident from monomer to dimer ratio, depends on the molar concentrations of MB, which is significantly higher in water compared to other formulations. MIX-based MB formulation effectively penetrates the dentinal tubules. Although, the affinity of MB for Enterococcus faecalis (gram positive) and Actinomycetes actinomycetemcomitans (gram negative) was found to be high in the water-based formulation, followed by MIX, the MIX-based formulation significantly enhanced the model substrate photooxidation and singlet oxygen generation compared to MB dissolved in other formulations. Finally, the efficacy of LAT is evaluated on biofilms produced by both organisms under in vitro and ex vivo conditions. A dual-stage approach that applies a photosensitization medium and an irradiation medium separately is tested. The MIX-based photosensitization medium in combination with dual-stage approach demonstrates thorough disinfection of the root canal with bacterial biofilms. This method will have potential application for root canal disinfection.