Two-photon excitation photodynamic therapy (TPE-PDT) is an emerging approach to treat a broad range of tumors and infection-associated diseases with three-dimensional spatial and temporal resolution and increased penetration depth (> 2 cm) in living tissues compared to UV- visible light. Moreover, the biological transparency of near-infrared (NIR) window beyond 700 nm is essential to prevent absorption by biological tissues and blood that considerably minimize laser scattering. Furthermore, the integration of energy donor moieties with superior two- photon absorption (TPA) capacity in photoactive nanomaterials can even further increase ROS formation upon Förster Resonance Energy Transfer (FRET) to co-localized two-photon sensitizer (PS) acceptor moieties. Among cross-linked NPs, periodic mesoporous organosilica (PMO) NPs are noteworthy nanomaterials for biomedical applications. They can integrate functional complex systems that can specifically reach the targeted cells or tissues. Moreover, fine mesopores provide permeability to ROS diffusion from embedded PSs for PDT. The development of two-photon responsive PMO NPs was achieved with the integration of a tetra-sulfide component to enhance their biodegradation by redox reaction with glutathione reduced form (GSH), a natural tripeptide present in high rates in bacteria environment and in cancer cells. A highly two-photon active three-branched fluorophore (F) and a porphyrin PS (P) were integrated in sol-gel synthesis as a FRET pair candidate for enhanced TPE-PDT alongside NP tracking ability by NPs intrinsic fluorescence properties. Fluorescence measurements of PMO NPs demonstrated efficient intraparticle FRET from the Fluorophore moieties possessing high two-photon absorption cross-section in the nearinfrared region to the porphyrin photosensitizer moieties within the hybrid frameworks. As a whole, the design strategies of mixed PMO nanoparticles allowed obtaining suitable materials for both anticancer and bioimaging two-photon excitation PDT. Their internalization imaging in breast cancer cells and epithelial-like cells (MDA cell line) as well as their toxicity upon two-photon excitation PDT were highlighted using confocal microscopy via fluorescence imaging