Methylene blue (MB) is a well-established and extensively studied photosensitizer for photodynamic therapy (PDT), since it can generate singlet oxygen with a high quantum yield upon irradiation within the phototherapeutic (600–950 nm) window. However, its activity can decrease due to the formation of dimers or higher aggregates, which can take place in an aqueous solution at relatively high concentrations. The incorporation of this molecule into a matrix can avoid this aggregation and increase its activity relative to PDT. Silica porous nanoparticles are chosen here as a matrix to host MB. The size and pore geometry are tuned in order to decrease MB leaching while maintaining good singlet oxygen generation and colloidal stability for further applications in nanomedicine. In addition, phenyl functions are grafted on the pores of the silica matrix in order to avoid MB aggregation, thereby increasing the activity of the photosensitizer in the singlet oxygen generation. DFT calculations give insight in the structure of the aggregation of the MB units, and the roles of water and organic environments are investigated through time-dependent calculations on UV-vis spectra.