Purpose: To evaluate the difference in cytotoxicity of soft denture lining materials depending on their component types. Materials and Methods: Ten commercially available soft denture lining materials (SDLM) consisting of five silicone-based materials and five acrylic-based materials were evaluated. For the MTT test, cured SDLM samples were extracted in a culture medium for 24 hours, and L-929 cells were incubated in the extracted medium for 24 hours. Cell viability was determined using a microplate reader and compared with those of the negative control, which were cultured in a culture medium without test material. Agar overlay test was performed for the cured SDLM samples according to International Organization for Standardization (ISO) 7405. Results: Among silicone-based lining materials, GC Reline Soft, Mollosil plus, and Dentusil showed a cell viability of 107.2% ± 4.5%, 102.3% ± 2.84%, and 93.0% ± 8.0%, respectively, compared with the control. Mucopren and Sofreliner Tough displayed significantly lower cell viability (86.4% ± 10.3% and 81.5% ± 4.3%, respectively) compared with the control (P .05). Among acrylic-based materials, Kooliner, Visco-gel, Soft liner, Dura Base, and Coe-Soft displayed cell viability of 99.2% ± 14.6%, 93.1% ± 9.5%, 89.1% ± 9.8%, 87.6% ± 7.9%, and 75.9% ± 15.7%, respectively, compared with the control. Dura Base and Coe-Soft displayed significantly lower cell viability compared to the control. However, for all tested materials, cell viability exceeded the requirement limit of 70% specified in ISO 10993-5. In the agar overlay test, all five silicone-based materials and acrylic-based Kooliner were ranked as "noncytotoxic." However, Visco-gel was ranked as "mildly cytotoxic," and Soft liner, Coe-Soft, and Dura Base were ranked as "moderately cytotoxic." Conclusion: When an acrylic-based soft denture lining material is used, the possibility of a cytotoxic effect should be considered.