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Abstract Background: Recent studies has shown that toxic effects of chlorhexidine (CHL) is not limited to bacteria but also noxious for a variety of cells including sperms, polymorphonuclears, macrophages, epithelial cells, erythrocytes and gingival fibroblasts. Objective: To evaluate cytotoxic effects of chlorhexidine on rat L929 fibroblast cell line and also determining the safest and most effective dose of this agent. Methods: L929 fibroblast cell cultures supplemented with FBS were treated with 0.2, 0.12 and 0.009% of chlorhexidine concentrations for 30 seconds, 1 minute and 5 minutes. Then, Media was removed and cells were washed with RPMI three times and were incubated in new culture media with MTT for 4 hours. Since chlorhexidine cytotoxicity affects mitochondrial dehydrogenase in viable cells, no MTT reduction and further formazan crystal formation occurs. The optical density of the color changes was detected using ELISA reader. Findings: CHL was cytotoxic at all concentrations and time intervals used in our study. ANOVA showed a lack of any significant difference in toxic effects of chlorhexidine at different concentrations and durations. Conclusion: Regarding the cytotoxicity of CHL at concentrations and durations much less than those in clinical application, conservative use of chlorhexidine is recommended. Also, additional studies on CHL to determine a safe and effective dose and duration are suggested. Keywords: Chlorhexidine, Fibroblast, Cytotoxicity, Gingiva

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