The aim of the present study is to evaluate and compare the cytotoxic effects of eight root canal sealers (BioRoot RCS, TotalFill BC Sealer, MTA Fillapex, Sealapex, AH Plus, EasySeal, Pulp Canal Sealer, N2) on immortalized human gingival fibroblasts over a period of 24, 48 and 72 hours. Immortalized human gingival fibroblast-1 HGF-1 (ATCC CRL-2014) were incubated. Root canal sealers were then placed into sterile, cylindrical Teflon moulds. The extraction was made eluting the sealers in cell culture medium. Cells (1 × 104) were seeded in each well of a 96-well plate and incubated for 24 h at 37°C. Cultures were then exposed to 100 ?L of the extracts medium. The percentage of viable cells in each well was calculated relative to control cells set to 100%. BioRoot RCS and TotalFill BC Sealer extracted for 24h showed no cytotoxic effect, while it was mild by using 48 and 72 h extracts. No cytotoxic effect was measured by using AH Plus medium eluted for 24 h, while it was moderate after 48 h and severe after 72 h. Pulp Canal Sealer, Sealapex and N2 showed moderately cytotoxic activity for all the extraction times. EasySeal and MTA Fillapex remained severely or borderline mildly cytotoxic for all the extraction times. In the present study only BioRoot RCS, TotalFill BC Sealer and AH Plus showed no cytotoxic effects at least in the first 24h. All the other sealers revealed moderately or severely cytotoxic activity during all the extraction times.