Objective: This study evaluated the antibiofilm efficacy of nanoparticle agents using photodynamic therapy. Methods: A total of Ninety dentin section blocks were used in this study. Dentin section blocks were prepared to be (4×4×1 mm) (Length× Width × thickness) respectively. Each dentin section was placed in 1.5 mL polypropylene tube filled with brain heart infusion (BHI) broth then underwent sterilization using autoclave at 121°C for 20 minutes and the sterilization was confirmed using bacterial count test. Cultivation of Standard strains of E. Faecalis ATCC 29212 and Staph. Epidermidis ATCC 12228 were prepared then inoculated on dentin section blocks and incubated for 3 weeks at 37°C in incubator (The medium was changed every 3 days). The specimens were grouped into 4 groups according to Final irrigant rinse were used: G1: final rinse using chitosan nanoparticles (CSnps)., G2: final rinse using Rose Bengal dye (RB), G3: final rinse using chitosan nanoparticles conjugated with Rose Bengal dye (CSRBnps) and G4: control group. Each group was activated using a diode laser 635 nm for 5 minutes at energy fluence 30 J/cm2. Then, the effect of tested final rinsing on multispecies biofilm was assessed with Confocal Laser Scanning Electron Microscope (CLSM) and data were statistically analyzed. Results: With regard to the antimicrobial effectiveness of tested irrigants, the highest mean percentage of dead bacteria was found in {CSRBnp (Group 3)} (88.38 ± 5.45) followed by {Csnp (Group 1)} (75.73 ± 5.15) followed by Rose Bengal dye (Group 2) (59.43 ± 8.14). The least mean percentage of dead bacteria was found in Control group (Group 4)} (7.25 ± 1.12). With a statistically significant difference between groups where (p<0.001). Conclusion: Functionalization of chitosan nanoparticles with Rose Bengal dye is a potent antibiofilm strategy for disinfection of the root canal system.