Aim: This study seeks to assess the cytotoxic impact of various bioceramic sealers, namely Well-Root ST, CeraSeal, and NeoSEALER Flo, in comparison to AH Plus sealer. The evaluation encompasses both the fresh and set states of these sealers, employing the MTT assay on human periodontal ligament fibroblasts. Materials and methods: Following the manufacturers' instructions, the four tested sealers were blended in a sterile environment and then placed into standardized plastic rings. Extracts derived from the tested sealers were applied to human periodontal ligament fibroblasts. The freshly mixed sealers were examined immediately after mixing, with their extracts prepared at that moment. On the other hand, the remaining specimens, designated as set specimens, were incubated in a humidified environment with 5% CO2 and 95% air at 37º C for 24 hours before extraction to create extracts of the tested sealers. The extracted material was then diluted with DMEM to achieve twelve distinct concentrations of each extract 50%, 25%, 12.5%, 6.25%, 3.12%, 1.56%, 0.78%, 0.4%, 0.2%, 0.1%, 0.05%, and 0.025%. The cytotoxicity of all root canal sealers was evaluated using the MTT assay, followed by the calculation of cell viability percentages. Results: AH Plus showed the highest toxicity followed by the NeoSEALER Flo then Well-Root ST and the least toxicity was CeraSeal. Conclusion: The assessed root canal sealers exhibited differing levels of cytotoxicity, and the rise in cell viability percentages was contingent on the concentration.