Aminolevulinic acid (ALA) based photodynamic antimicrobial strategy can provide good antimicrobial effects and be used for medical applications. The aim of this study was to apply this strategy to Mineral Trioxide Aggregate (MTA), which is commonly used as a filling material for root endings and by doing so, to increase the bactericidal capability of MTA, as well as to investigate its characterization, cytocompatibility, and odontogenic differentiation potential. MTA is known to be a derivative of calcium silicate (CS). In this study, MTA specimens with or without ALA and light treatment were prepared. Diametral tensile strength values (DTS), setting durations, X-ray diffraction (XRD) spectra, apatite-mineralization, and antimicrobial abilities of the MTA, were also analyzed. Human dental pulp cells (hDPCs) can proliferate into the newly formed matrix and differentiate into odontoblasts to reinforce and strengthen the root. Levels of hDPCs proliferation and its odontogenic capabilities when cultured on MTA with ALA and light treatment, and the percentages of cells existing in the various cell cycle stages, were further evaluated in this study. The results indicated that MTA added ALA with light treatment had greater antibacterial ability and cytocompatibility, compared to MTA alone. A higher percentage S phase of the cells cultured on MTA added ALA with light treatment was observed. Furthermore, hDPCs cultured on MTA added ALA with light treatment had the highest expression levels of the odontoblastic differentiation markers. ALA has great antimicrobial efficiency and is a potential material for future medical applications. ALA-based photodynamic antibacterial strategy applied in the MTA has great antibacterial ability, cytocompatibility, and odontoblastic differentiation potential, and can facilitate the development of root canal treatment.