AimTo assess the effects of octenidine dihydrochloride (OCT) on eukaryotic cells and the cytotoxicity of OCT associated with sodium hypochlorite – NaOCl (NaOCl/OCT).MethodologyL929 fibroblasts and human osteoblast‐like cells (Saos‐2) were exposed to 0.1% OCT, 2% CHX, 2.5% NaOCl, 5.25% NaOCl and mixtures of 5.25% NaOCl and 0.1% OCT (NaOCl/OCT) at 90 : 10, 80 : 20 and 50 : 50 ratios. Cell viability was assessed by methyl‐thiazol‐tetrazolium (MTT) and neutral red (NR) assays; type of cell death, by flow cytometry; cytoskeleton, by actin and α‐tubulin fluorescence; and alkaline phosphatase (ALP) activity, by thymolphthalein release. The data were analysed by two‐way ANOVA and Bonferroni tests (α = 0.05).ResultsMTT and NR assays revealed that 0.1% OCT had the lowest cytotoxicity (P < 0.05), followed by 2% CHX (P < 0.05). The 2.5% NaOCl, NaOCl/OCT 80 : 20 and NaOCl/OCT 50 : 50 solutions had intermediate cytotoxicity. NaOCl 5.25% and NaOCl/OCT 90 : 10 had the highest cytotoxicity (P < 0.05). The OCT group had a higher percentage of viable cells than the NaOCl and CHX groups (P < 0.05), and induced apoptosis at higher doses. The cytoskeleton alterations were observed at 0.12%, 0.6% and 2.02% for the NaOCl, CHX and OCT groups, respectively. The solutions did not induce ALP activity.ConclusionOctenidine dihydrochloride was less cytotoxic, induced apoptosis at higher doses, caused few changes in the cytoskeleton and did not induce alkaline phosphatase activity. In addition, octenidine dihydrochloride reduced the cytotoxicity of 5.25% NaOCl when combined at 20 and 50%.