Aim The purpose of this study was to determine the cytotoxicity of three different types of root canal sealer on human periodontal ligament (PDL) cells and a permanent hamster cell line (V79 cells). Methodology Set specimens from two resin based sealers (AH26 and AHPlus), three zinc oxide-eugenol-based sealers (Canals, Endomethansone and N2) and one calcium hydroxide-based sealer (Sealapex) were eluted with culture medium for 1, 2, 3 and 7 days. Cytotoxicity was judged using tetrazolium bromide reduction assay on human primary PDL cells and V79 cells derived from a Chinese hamster. Results The results showed that elutes from resin-based, zinc oxide-eugenol-based, and calcium hydroxide-based sealers were cytotoxic to primary human PDL cultures and V79 cells. Calcium hydroxide-based sealer was the least toxic sealer amongst the chemicals tested in both cultures. The cytotoxic response decreased in an order of N2 > Endomethansone > AH26 > AHplus > Canals > Sealapex. Conclusions The sensitivity of toxicity depended on the materials tested and the cell culture system used. Thus, the use of both permanent and primary cells is recommended for screening of the cytotoxic effects of root canal sealers. In addition, the results confirmed that root canal sealers constantly dissolve when exposed to an aqueous environment for extended periods, possibly causing moderate or severe cytotoxic reactions. Use of calcium hydroxide-based material as a root canal sealer initially may result in a more favourable response to periradicular tissues.