Objectives: This study evaluated the cytotoxicity of different thermoplastic materials and the impact of thermoforming on their cytotoxicity. Materials and methods: Four 3D thermoplastic materials were tested before and after thermoforming: Zendura A (Bay Materials), Zendura FLX (Bay Materials), Erkoloc-pro (Erkodent), and CA Pro + (Scheu Dental). The samples were stored in artificial saliva and incubated at 37 °C for 14 days to simulate conditions in the oral cavity during one stage of clear aligner treatment. Then, the saliva was diluted with a complete culture medium at three concentrations: c1=10%, c2=20% and c3=30%. Cytotoxic activities of the materials were evaluated after incubation for 4h, 24h, 48h, and 72h by using the CCK-8 assay on human oral fibroblast cells (Innoprot, REF: P10868). Results: The mean measured values of metabolically active cells below 70% of the control were for c3 of non-thermoformed Zendura A and Erkoloc-pro, and thermoformed Zendura A, after 72-hour incubation. The analysis showed differences between non-thermoformed: Zendura A and Erkoloc-pro and Zendura FLX and CA Pro +, thermoformed: Zendura A and CA Pro +, and Zendura FLX and CA Pro + at certain conditions. Differences between concentrations (c2 vs. c3 and c1 vs. c3) in the non-thermoformed group were found. There were no statistically significant differences in cytotoxicity between non-thermoformed and thermoformed groups. Conclusions: Cytotoxicity was observed in non-thermoformed and thermoformed materials, with significant differences between concentrations of the same material. However, the thermoforming process did not impact the cytotoxicity of clear aligner materials.