Objective: To analyze effect of NaOCl + 2% quaternary ammonium silane (QAS)-containing novel irrigant against bacteria impregnated inside the root canal system, and to evaluate its antimicrobial and mechanical potential of dentine substrate. Methods: Root canal was prepared using stainless steel K-files™ and ProTaper™ and subjected to manual and ultrasonic irrigation using 6% NaOCl + 2% CHX, 6% NaOCl + 2% QAS and saline as control. For confocal-microscopy, Raman spectroscopy and SEM analysis before and after treatment, Enterococcus faecalis cultured for 7 days. Raman spectroscopy analysis was done across cut section of gutta percha/sealer-dentine to detect resin infiltration. Indentation of mechanical properties was evaluated using a Berkovich indenter. The contact angle of irrigants and surface free energy were evaluated. Mineralization nodules were detected through Alazarin red after 14 days. Results: Control biofilms showed dense green colonies. Majority of E. faecalis bacteria were present in biofilm fluoresced red in NaOCl + 2% QAS group. There was reduction of 484 cm−1 Raman band and its intensity reached lowest with NaOCl + 2% QAS. There was an increase in 1350–1420 cm−1 intensity in the NaOCl + 2% CHX groups. Gradual decrease in 1639 cm−1 and 1609 cm−1 Raman signal ratios were seen in the resin-depth region of 17 μm>, 14.1 μm> and 13.2 μm for NaOCl + 2% QAS, NaOCl + 2% CHX and control groups respectively. All obturated groups showed an intact sealer/dentine interface with a few notable differences. 0.771 and 83.5% creep indentation distance for NaOCl + 2% QAS ultrasonic groups were observed. Highest proportion of polar component was significantly found in the NaOCl + 2% QAS groups which was significantly higher as compared to other groups. Mineralized nodules were increased in NaOCl + 2% QAS. Significance: Favorable antimicrobial and endodontic profile of the NaOCl + 2% QAS solution might suggest clinical use for it for more predictable reduction of intracanal bacteria.