The aim of this study was to compare the cytotoxicity of calcium silicate-based endodontic sealer, Bio-C® Sealer, with other calcium silicate-based sealers: BioRoot™ RCS, one silicon-based sealer combined with calcium silicate particles: GuttaFlow® Bioseal, one resin MTA-based root canal sealer: MTA Fillapex®, and an epoxy resin-based sealer: AH Plus®.NIH 3T3 fibroblasts were cultured and sealers extracts were obtained. Cytotoxicity was evaluated by the MTS assay and the optical densities of the solutions were measured with a microplate reader. This study was designed with one sample for each control group and n=10 for each treatment group of the different sealants. The results were classified according to the degree of cell viability and underwent statistical analysis with the ANOVA test (p<0.05). The samples were examined under an inverted microscope to evaluate the effect of each sealer on fibroblast cell morphology.Cells incubated with GuttaFlow® Bioseal extract showed the highest cell viability without statistically significant differences with the control group. BioRoot™ RCS and Bio-C® Sealer showed moderate (tending to slight) cytotoxicity and both AH Plus® and MTA Fillapex® showed severe cytotoxicity in comparison with the control group (p<0.05). AH Plus® and MTA Fillapex® were not significantly different from one another; nor was BioRoot™ RCS from Bio-C® Sealer. Microscope examination found that fibroblasts in contact with GuttaFlow® Bioseal and Bio-C® Sealer presented the most similar aspects to the control group both in terms of number and shape.Bio-C® Sealer showed moderate (tending to slight) cytotoxicity compared with the control group, GuttaFlow® Bioseal showed no cytotoxicity, BioRoot™ RCS moderate-slight cytotoxicity and AH Plus® and MTA Fillapex® severe cytotoxicity. Key words:Biocompatibility, calcium silicate-based endodontic sealers, cytotoxicity, endodontic sealer.