A bstract Aim To evaluate and compare the antimicrobial efficacy of 2.5% sodium hypochlorite (NaOCl) and Aquatine Endodontic Cleanser (EC) with and without laser-activated disinfection against Enterococcus faecalis in the root canals of primary teeth. Materials and methods A total of 45 human primary teeth were selected and inoculated with E. faecalis and divided into three groups as per intervention. In group I, irrigation was done using 2.5% NaOCl, in group II using Aquatine EC solution, and in group III with Aquatine EC activated by 810 nm diode laser. Results Intragroup comparisons showed a decrease in colony-forming units in all three groups. Intergroup comparisons showed a statistically significant difference between group I and group II ( p = 0.024) and between group I and group III ( p = 0.03). Conclusion Aquatine EC showed maximum antimicrobial efficacy under laser activation. Clinical significance Aquatine EC can be considered a suitable alternative to NaOCl in light of its known toxic effects. How to cite this article Kodical S, Attiguppe P, Siddalingappa RO, et al. Laser Activation of Aquatine Endodontic Cleanser: A Novel Approach to Root Canal Disinfection. Int J Clin Pediatr Dent 2022;15(6):761-763.