Introduction: The aim of this study was to evaluate the effect of functionalized nanoparticle photodynamic therapy on Nano hardness of root dentin Methodology: Fifty single rooted lower premolars were decoronated and sectioned into two halves. Then the samples were embedded horizontally in to the acrylic resin to expose the dentin surface. Baseline nanohardness was done at midroot level using a Nanohardness tester. Exposed dentin surfaces were immersed in the following irrigating solutions • Group 1: 1 ml of Chitosan conjugated Rose Bengal (CSRB-np) activated with 540 nm for 10 min • Group 2: 1 ml of Methylene blue loaded poly lactic acid (PLGA-MB np) activated with 660 nm for 10 min • Group 3: 5 ml of 5.25 % sodium hypochlorite for 1 min + 1 ml of 17 % EDTA for 1 min • Group 4: 1 ml of 2.5 % sodium hypochlorite for 10 min + 1 ml of 17 % EDTA for 1 min + 1 ml of 2% chlorhexidine for 1 min. • Group 5: 1 ml of 0.9 % Saline for 10 min. Post treatment nanohardness testing was done and results were analyzed statistically Results: In general, all the samples in their respective groups had significant change in nanohardness following immersion in irrigant solutions except in NaOCl + EDTA and saline group. CSRB-np and PLGA-MBnp showed increased nanohardness (P = 0.005 and P = 0.007 respectively). Whereas NaOCl + EDTA + CHX showed decrease in nanohardness (P = 0.04). With regards to Modulus of elasticity (MOE), CSRB-np showed significant difference (P = 0.002) compared to the other groups. MOE increased in CSRB-np and PLGA-MBnp while it decreased in all the other groups. Conclusion: In this study, the improvement of nanohardness and modulus of elasticity following the immersion of root dentin in CSRB-np solution was demonstrated © 2020 Elsevier B.V.