BACKGROUND The aim of this study was to evaluate the cytotoxicity of a new nano zinc-oxide eugenol (NZOE) sealer in comparison with AH-26 and Pulpdent root canal sealers. METHODS The L929 mouse fibroblast cells were cultivated and incubated for 24, 48 or 72 h with different dilutions (1/1, 1/2, 1/4, 1/8, 1/16 and 1/32) of culture media previously exposed to either of the test sealers naming NZOE, AH-26 or Pulpdent. At the end of incubation period, the effect of sealers on cell viability was evaluated using Mosmann's Tetrazolium Toxicity (MTT) colorimetric assay. The data was compared using the one-way analysis of variance (ANOVA) followed by the Tukey's post hoc test for multiple comparisons. RESULTS After 24, 48 or 72 h, both NZOE and Pulpdent sealers inhibited cell viability at 1/1, 1/2 and 1/8 dilutions. Within the 24 and 48 h, the AH-26 sealer reduced the cell viability at all dilutions except the 1/32 solution; however after 72 h even the 1/32 dilution was cytotoxic. CONCLUSIONS The biocompatibility of the nano zinc-oxide eugenol sealer was comparable to Pulpdent sealer and lower than AH-26.