(1) Background: Bacterial reinfection and root fracture are the main culprits related to root canal treatment failure. This study aimed to assess the utility of quercetin solution as an adjunctive endodontic irrigant that strengthen root canal dentin with commitment anti-biofilm activity and bio-safety. (2) Methods: Based on a noninvasive dentin infection model, dentin tubules infected with Enterococcus faecalis (E. faecalis) were irrigated with sterile water (control group), and 0, 1, 2, 4 wt% quercetin-containing ethanol solutions. The live and dead bacteria proportions within E. fae-calis biofilms were analyzed using confocal laser scanning microscopy (CLSM). Elastic modulus and hydroxyproline release and X-ray photoelectron spectroscopy (XPS) characterization was tested on irrigant-treated demineralized dentin to evaluate irrigants’ biostability. The cytotoxicity of irrigants was tested by CCK-8 assay. (3) Results: Quercetin increased the proportion of dead bacteria volumes within E. faecalis, and improved the flexural strength of dentin collagen com-pared to control group. The XPS characterization revealed an increase in C-O peak area under both C1s and O1s narrow-scan spectra. The CCK-8 assay confirmed no cytotoxicity of quercetin solutions. (4) Conclusions: Quercetin exhibited anti-biofilm activity, collagen-stabilizing effect as well as cytocompatibility, supporting quercetin as a potential candidate for endodontic irrigant.