Background: This study aimed to assess the cytotoxicity of four commonly used endodontic sealers namely AH Plus, AdSeal, Endoseal MTA, and GuttaFlow Bioseal against human gingival fibroblasts (HGFs). Methods: After culturing the HGFs, they were exposed to the respective sealers in set form and in five different weights, after sterilization. The cytotoxicity of the sealers was evaluated after 1, 3 and 7 days using the methyl thiazolyl tetrazolium (MTT) assay. Data were analyzed by repeated measures ANOVA. Results: After 24 h, all sealers showed low cytotoxicity. However, all sealers in 250 mg and 500 mg weights showed significantly higher cytotoxicity than the negative control group at 72 h, and 7 days (P<0.05) except for AdSeal in 80 mg weight (P>0.05). AH Plus was significantly more cytotoxic than other sealers at 3 and 7 days (P<0.05) while AdSeal had the closest results to the negative control group, and showed significantly higher biocompatibility than other sealers in 250 mg concentration. Conclusion: AdSeal showed the highest biocompatibility while AH Plus had the highest cytotoxicity among the tested sealers. Thus, its application may delay the healing of periapical lesions.