Background: Endodontic treatment of various forms of pulpitis with variations of root canal system anatomy should be performed with high quality. The use of various antibacterial agents is aimed at maintaining the success of endodontic treatment. The aim of this study was to evaluate the penetration and fixation of the nano-silver solution on the dentinal surface during endodontic treatment. Materials and methods: the study was carried out on 70 extracted single-rooted teeth, randomly divided into two groups. In the teeth of the first group, the smear layer was removed after canal preparation with 17% EDTA solution; in the second group, the smear layer was not removed. In both groups, for the final treatment of the canal, a colloidal 1% solution of нанo серебра nanosilver was used. Samples were cut and prepared for analysis using micro-CT, scanning electron microscopy (SEM), X-ray microanalysis and energy dispersive spectrometry (elemental mapping). Results: in 100% of cases in groups of teeth with a preserved smear layer, the ability of a 1% colloidal solution of nanosilver with particles of 1–2 nm to be fixed on dentin with a removed and preserved smear layer and to leave a film on the dentinal surface was established. In the samples with removed smear layer, silver was found in 73.5% of cases. Conclusion: The nano-silver solution with a particle size of 1–2 nm proved its ability to penetrate the dentinal surfaces and create a final film covering the dentinal surface of the root canal before applying the sealer.