Introduction: The aim of the present study was to evaluate the in vitro cytotoxicity of three endodontic sealers (TotalFill BC, Gutta Flow2 and AH Plus) on WI-38 cell line human. As a reference, AH Plus was compared with the more recent endodontic sealers regarding cell viability of fibroblast cells (WI-38). Methods: Biological testing was carried out in vitro on WI-38 cells. Cell viability assay was performed by using eluates from each endodontic sealer and dispensed to pre-cultured WI-38 cell line. The effect was assessed by MTT cell proliferation assay and ELISA reader after 24 hours and 72 hours of exposure and various concentrations of (0.5, 1, 2, 4, 8, 16, 32, 64, 125, 250 and 500mg/ ml). Statistical differences were assessed by analysis of variance and Tukey test (P < .05). Results: TotalFill BC, Gutta Flow2 and AH Plus sealer eluates for 24 hours showed no cytotoxic effect, while it was increased by increasing the concentration for 72 hours' measurement. Statistically there is no significant difference among the tested sealer materials; TotalFill BC, Gutta Flow2 and AH Plus. Conclusions: In the present study TotalFill BC, Gutta Flow2 and AH Plus sealer eluates showed no cytotoxic effects at least in the first 24 hours. But they exhibited moderately or severely cytotoxic activity at 72 hours of measurement at high concentration.