Current studies in photodynamic therapy (PDT) against cancer are focused on the development of new photosensitizers (PSs), with higher phototoxic action. The aim of this study was to compare the therapeutic efficiency of tri-cationic meso-substituted porphyrin derivatives (Tri-Py + -Me-PF, Tri-Py + -Me-Ph, Tri-Py + -Me-CO 2 Me and Tri-Py + -Me-CO 2 H) with the well-known tetra-cationic T 4 PM. The phototoxic action of these derivatives was assessed in human colon adenocarcinoma cells by cell viability, intracellular localization and nuclear morphology analysis. In the experimental conditions used we determined that after light activation -PF, -Ph and -CO 2 Me cause a more significant decline of cell viability compared to -CO 2 H and T 4 PM. These results suggest that the nature of the peripheral substituent influences the extent of cell photodamage. Moreover, we have demonstrated that PS concentration, physicochemical properties and further light activation determine the PDT response. All porphyrins were clearly localized as a punctuated pattern in the cytoplasm of the cells, and the PDT scheme resulted in apoptotic cell death after 3 h post-PDT. The tri-cationic porphyrin derivatives Tri-Py + -Me-PF, Tri-Py + -Me-Ph and Tri-Py + -Me-CO 2 Me showed a promising ability, making them good photosensitizer candidates for oncological PDT.