Aim: The aim of this study is to evaluate the antibacterial effect of Salvadora persica nanoparticles and 2.6 % NaOCl as root canal irrigants against E. Feacalis bacterial biofilm using confocal laser microscopy. Methods and Materials: This study was performed on 48 recently extracted single canaled mandibular premolar teeth that were divided randomly into three groups. E. Feacalis bacterial incubation into root canals for three weeks to ensure biofilm maturation. The specimens were stained with the live/dead technique and evaluated using a confocal laser scanning microscope. Comparison between different groups was performed by using One Way ANOVA test which revealed significant difference between all groups regarding percentage of dead cells as P<0.05, followed by Tukey`s Post Hoc test 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 represented an efficient and natural root canal irrigant against E.faecalis biofilm.