Purpose: The purpose of this study was to assess the cytotoxic effect of different concentrations of Chitosan Nanoparticles (CNPs) and Propolis Nanoparticles (PNPs) on periodontal ligament stem cells using MTT assay. Materials and Methods: Immature, impacted third molars were used to isolate stem cells. Cells were seeded in 96-well culture plates. Groups were divided into six experimental groups: Group I: 17% EDTA, Group II: 1% propolis NPs, Group III: 2% propolis NPs, Group IV: 1% chitosan NPs, Group V: 2% chitosan NPs, Group VI: cultured cells not receive any material. Cytotoxic effect of these materials on stem cells was evaluated using MTT assay at 24, 72,120 hours and 14 days. Results: In all assay intervals; 2%CNPs group showed the statistically significant highest median value and control group showed the statistically significant lowest median values. After 24 and 72 hours; there was no statistically significant difference between 2% PNPs and 1% CNPs groups followed by 1% PNPs group. Both EDTA and control groups; showed the statistically significantly lowest median values with no statistically significant difference between them. After 120 hours; there was no statistically significant difference between 2% PNPs and 1% CNPs groups in median value followed by 1% PNPs then EDTA groups. After 14 days; there was no statistically significant difference between 1% PNPs, 2% PNPs and 1% CNPs groups in median value followed by EDTA group. Conclusion: The ability of 2% Chitosan NPs to promote periodontal ligament stem cell viability makes them a promising irrigating solution. The cell viability of 2% Propolis NPs was comparable to that of 1% Chitosan NPs, which was better than EDTA.