The aim of this study was to evaluate the cytotoxic effects of five different resin-based root canal sealers: EndoREZ, Epiphany SE, EZ-Fill, MMSeal and AHPlus. Set materials were extracted in culture medium and cytotoxicity was determined in two cell lines, human osteosarcoma cell line (Saos-2) and mouse skin fibroblast cells (L929). The cells were incubated in contact with elutes for 24 h. The cell mitochondrial activity was evaluated by the methylthiazole tetrazolium assay. Results with demonstrated that all sealers showed a reduced vital cell number in comparison with the control group (P < 0.05). For L929 cells, the ranking of the most to the least toxic material was: EZ-Fill (12.0%) = EndoREZ (12.1%) = AHPlus (12.4%) > MMSeal (44%) = Epiphany SE (46.2 %). For Saos-2 cells revealed that cell survival with extracts of EndoREZ, Epiphany SE, EZ-Fill, MMSeal and AHPlus was 33.9%, 32.9 %, 33.1%, 35.3% and 34.6%, respectively, all tested sealers showed moderate cytotoxicity. Based on the results obtained from the present study, all tested resin-based sealers appear to show toxicity potential to both cells in spite of different toxicity degrees. Therefore, better sealers need to be developed with acceptable biological properties for root canal filling.