Three dimensional (3D) printing materials are widely used in dental applications, but their biocompatibility and interactions with human cells require evaluation. This study aimed to identify materials meeting biocompatibility, mechanical strength, and tissue-forming requirements for safe dental applications. We assessed the cytotoxicity of resins and thermoplastic filaments in human HaCaT keratinocytes, gingival fibroblasts (hGFs), and stem cells from human exfoliated deciduous teeth (SHED) using PrestoBlue assays. Three resins, including two types of surgical guide resins, exhibited strong cytotoxicity after 4-72 h, while 2 h exposure to an FDA-approved surgical guide resin did not affect SHED cell viability. In contrast, six thermoplastic filaments showed no significant cytotoxicity even after 72 h. Among these, polycarbonate-acrylonitrile butadiene styrene (PC-ABS) demonstrated excellent toughness, heat resistance, and surface quality at a low cost. SHED cells cultured on PC-ABS dishes and micro bone structures showed strong proliferation and osteogenic potential. Culture inserts made of PC-ABS also supported the growth of HaCaT keratinocytes and the hGFs formed gingival tissue, which was superior to that formed on commercially available PET inserts. In conclusion, PC-ABS is a promising 3D printing material for dental applications due to its biocompatibility, ability to promote osteogenesis, and support for gingival tissue formation, with no observed cytotoxicity.