Aim: The study was done to evaluate the antibacterial effect of a new formula of Salvadora persica roots Nanoparticles as compared to sodium hypochlorite irrigating solution. Methods: A total of 20 recently extracted single-rooted, with single root canal were selected to be used in the study. Decapitation of the selected teeth for 18 mm length standardization was done. The root canals were initially instrumented using manual instrumentation till reach file size 25 for apical preparation. Sterilization of the samples was done using gamma rays. Following sterilization, the samples were divided into two groups (n=10) according to the tested irrigating solution used as follow; Group H: For Salvadora persica roots nanoparticles. Group S: For 2% sodium hypochlorite. The chemo-mechanical preparation was accomplished using Revo-S rotary system in a crown-down technique with the file sequence that was recommended by the manufacturers. Pre and post-irrigation counting of the E. Fecalis was done by determining the Colony Forming Unit. Results: The highest mean value of percentage changes in bacterial reduction was found in (Group S) (95.99%) and the least bacterial reduction percentage was found in (Group H) (82.83% with no statistically significant difference between both groups (P-value=0.068). Conclusion: Under the condition of this study, the new formula of Salvadora persica roots nanoparticles is a promising irrigating solution of herbal origin instead of the synthetic irrigating solution.