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Dental materials should be biocompatible, in order to prevent any adverse effects on the surrounding tissue caused by direct contact. The ideal root end filling material must have certain characteristics, including biocompatibility, satisfying marginal sealing quality, ability to permit or induce repair of alveolar bone, promote periapical healing and antimicrobial activity. In this study, the cytotoxicity of different materials (amalgam, MTA and Biodentine) was evaluated on a permanent fibroblast cell lines (MRC-5 and L929). The cytotoxicity of all three types of materials was investigated using standard biocompatibility tests: DET, MTT and agar diffusion test. MTT test showed that after 24 and 48 hours in both cell cultures, Biodentine had the largest percentage of citotoxicity. The lowest percentage of cytotoxicity showed MTA in both groups. After 72 h in both cell lines, the highest percentage of cytotoxicity had amalgam. The lowest percentage of cytotoxicity showed MTA in both groups. Observing the results of Agar diffusion test, there was no any discoloration detected, neither lysis of cells under the disc. Biocompatibility tests showed high level of cell compatibility of all three tested materials. © 2014 American Scientific Publishers All rights reserved.