This study evaluated the effect of the incorporation of bioactive nanofibers in desensitizing agents on dentin permeability. Sixty disks of dentin were randomly distributed in four groups (n = 15). Distribution was based on the desensitizing agents, fluoride varnish and self-etching adhesive, and the presence of nanofibers: C (self-etching adhesive Clearfil SE Bond), CN (Clearfil SE Bond with 1% nanofiber), D (Duraphat varnish), and DN (Duraphat varnish with 1% nanofiber). Dentin permeability was determined using hydraulic conductivity. For a qualitative analysis, confocal laser microscopy and scanning electron microscopy were performed. The C group showed the lowest hydraulic conductance (Lp%) (89.33), while the DN group showed the highest Lp% (116.06). No statistical significance was observed in the Lp% values in all groups after the treatment and 6% citric acid challenge (p > 0.239). In the images, the CN group presented a higher superficial and intratubular deposition. In addition, this group presented a more homogeneous dentin surface and wide occlusion of dentinal tubules than the other treatments. Despite there being no statistical differences among the treatments employed, the images showed that the CN group presented a higher surface and intratubular deposition compared to the other treatments, even after the acid challenge.