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<p><strong>Background &amp; Objective: </strong>Chlorhexidine solution is one of the widely used mouth antiseptic liquid that prevents teeth tissue damage and also has application as a root canal antiseptic. In this study, cytotoxicity of 2% chlorhexidine solution is compared with another root canal antiseptic, calcium hydroxide powder.</p><p><strong>Materials and Methods: </strong>Cell cytotoxicity of both chemicals was assessed on cultured L929 fibroblastic cell line for 1,</p><p>12, 24, 48 and 72 hours using MTT assay (Methyl tetrazolium bromide assay). Untreated L929 cells were used as a negative control group. MTT results were recorded by ELISA reader and analyzed using one-way ANOVA statistical tests.</p><p><strong>Results: </strong>Cytotoxicity of studied chemicals showed significant difference in various dilutions and times (1, 12, 24, 48 and 72 h). The highest cytotoxic effect of 2% chlorhexidine solution was observed in concentration of 0.016% for 72 h. Treatment of cells with 0.016% of 2% chlorhexidine liquid and calcium hydroxide powder for 72 hours showed 80% and 45% cytotoxicity, respectively.</p><p><strong>Conclusions: </strong>Cytotoxicity of calcium hydroxide is significantly less than 2% chlorhexidine liquid and then application of calcium hydroxide powder as root canal antiseptic is recommended.</p>