Aim: This study aimed to assess the antibacterial efficacy of Salvadora persica nanoparticles and 2.6 % sodium hypochlorite as root canal irrigants against E. Feacalis biofilm using confocal laser microscopy. Methods and Materials: 48 single canaled mandibular premolar teeth that were recently extracted and randomly split into three groups were used for this study. To guarantee the development of bacterial biofilms, E. fecalis bacteria are incubated in root canals for three weeks. A confocal laser scanning microscope was utilized to evaluate the specimens after they had been stained using the live/dead technique. One Way ANOVA was used to compare the various groups, and the results showed that there was a significant difference (P<0.05) in the percentage of dead cells across all groups. Tukey's Post Hoc test was then used to account for multiple comparisons. Results: Regarding percentage of dead cells Salvadora persica nanoparticles group (52.17 % ± 2.31) was significantly the highest, while control group (1.47 % ± 0.24) was significantly the lowest. Conclusion: Salvadora persica nanoparticles proved to be an effective and all-natural root canal irrigant against the biofilm of E.faecalis.