Introduction: Root canal infections are multifactorial and are caused due to a diverse species of organisms present in the oral cavity and the root canal. Sodium hypochlorite has long been the gold standard for irrigation. However, there are several significant disadvantages to using sodium hypochlorite.Chitosan is a natural biopolymer having antimicrobial activity and has all the properties of an ideal irrigant / sodium hypochlorite without its inherent drawbacks.The aim of this study was to evaluate the effect of Sodium hypochlorite, plain chitosan with chlorhexidine, and chitosan nanoparticles with chlorhexidine on the L929 fibroblast cell line. Materials and Method: The chitosan was obtained and plain chitosan with chlorhexidine, chitosan nanoparticles with chlorhexidine was prepared. For cell viability assay, cells were seeded onto 96-well plates at a concentration of 5×103 cells/well, followed by the addition of test samples of various concentrations prepared in cell culture media. MTT assay was done which measures the reduction of yellow 3-(4,5dimethiazol-2-yl)- 2, 5- diphenhydramine tetrazolium bromide(MTT) by mitochondrial succinate dehydrogenase. The effect of test samples (25-100μg/ml) on cell growth inhibition will be assessed as percentile viability, where vehicle-treated cells will be taken as 100% viable. Results: Statistical analysis was done using One-way Anova and a post hoc test. p<0.05 was considered as significant. P<0.001 as compared with negative control. Chitosan nanoparticles with chlorhexidine at different concentrations are P<0.001 as compared with sodium hypochlorite. Conclusion: Chitosan nanoparticles with chlorhexidine had less cytotoxic effect based on its concentration compared to sodium hypochlorite. Hence it can be used as an alternative endodontic irrigant.