Aim : The aim of the present study was to compare In-Vitro three newcombinations of root canal irrigants for their antimicrobial activity, debris analysis and, their effect on debris and the smear layer removal namely: 6% sodium hypochlorite with surface modifiers, 2% Chlorhexidine gluconate withsurface modifiers, and Tubulicid Plus.Methodology : Enterococcus faecalis (batch no ATCC 29212) thawedand incubated under anaerobic conditions then cultivated in nutrient agar. The harvested bacterial colonies are then spectrophotometrically calibrated to 5.9 x 104 (CFU/mL-1). Forty one specimens instrumented conventionally, reachingmaster apical file size #55. Twenty μL of the bacterial culture transferred to the canal lumen using sterile micropipette. All specimens then incubated for 72 hours at 37 ˚C. Stereomicroscope with a digital camera used to examine thedebris along apical and middle third and analyzed by image J. Three random specimens from each group were examined under ESEM at the middle and apical third at 600-X and 1200-X magnification at 30 Kv.Results : Intracanal sample collection by paper points showed the highest significant bacterial count was produced by negative control (saline) followedby positive control (2.5% NaOCl) followed by Tubulicid Plus. Meanwhile; Chlor-XTRA and CHX-Plus both produced an insignificant effect in decreasing the bacterial count between each other, but significant compared to 2.5% NaOCl(control) and Tubulicid Plus. Outflow sample collection in sterile containers showed only the negative control group produced significant higher values ofbacterial count. Debris analysis in the middle and apical segments showed Tubulicid Plus produced the highest significant reduction in debris area followed by Chlor-XTRA and CHX-Plus followed by 2.5% NaOCl , negative control produced the highest significant value for the remaining debris. Layer score in the middle and apical segments, The Kruskal–Wallis nonparametric one-way ANOVA showed a significant differences in smear score (at p=0.005) for the middle area and (p=0.002) for the apical area rejecting the null hypothesis that the distribution of smear scores is the same acrosscategories of irrigant at the middle and apical area.Conclusion: Chlor-Xtra and CHX-Plus has better antibacterial propertieswhen compared to Tubulicid Plus against Enterococcus Faecalis. Tubulicid plus has better effect on debris reduction followed by Chlor-XTRA. Tubulicid Plus is the most effective irrigant to remove smear layer than any other irrigant used in this study