The aim of this study was to evaluate the biocompatibility of epoxy-resin-based AHPlus, salicylate-based MTA-Fillapex and calcium silicate-based iRootSP root canal sealers. Cytotoxicity was assessed by XTT test. The extracts from sealers of different setting times were serially diluted. Cell viability was calculated as the percentage of the control group (100%). The optimal concentration of each sealer was used at genotoxicity test, and micronuclei formations were detected. Statistical analyses were done by using Kruskal–Wallis and Dunn post hoc test with Bonferroni correction. AHPlus and MTA-Fillapex showed the lowest percentage of cell viability at higher concentrations (1:1, 1:2, 1:4), especially at first 12 h. iRootSP showed higher viability at all concentrations and times than AHPlus and MTA-Fillapex. At genotoxicity assay, AHPlus increased the number of micronuclei. MTA-Fillapex slightly induced micronucleus formation (not significant) and iRootSP was not increased. In conclusion, calcium silicate-based iRootSP had lowest cytotoxic and genotoxic potential and can be considered as a highly biocompatible material.