Background <p>This study evaluated the effects of dentifrices on dentin permeability, surface roughness, dentinal tubule occlusion, and dentin chemical composition after in-office desensitizing treatment and an erosive–abrasive challenge.</p> Methods <p>Human dentin specimens were treated with fluoride varnish and randomly allocated to five groups according to dentifrice used: fluoride-free (WF), sodium fluoride (NaF), fluorocalcium phosphosilicate (FCPS), arginine + calcium carbonate (AC), and NovaMin^® (NM). Specimens were subjected to a 5-day erosive–abrasive cycling protocol. Dentin permeability was assessed by hydraulic conductance at baseline (T0), after varnish application (T1), and after cycling (T2). Surface roughness and tubule morphology were evaluated using CLSM, and chemical composition was analyzed by ATR-FTIR. Data were analyzed using parametric and nonparametric tests (α = 0.05).</p> Results <p>Fluoride varnish reduced dentin permeability in all groups (T1 vs. T0; <i>p</i> &lt; 0.05). After erosive-abrasive challenge, permeability increased in all groups except FCPS. No differences among dentifrices were observed for permeability (<i>P</i> &gt; 0.05) or surface roughness (<i>P</i> ≥ 0.33). Morphological analysis showed lower tubule number, area, and perimeter in FCPS and NM groups (<i>P</i> &lt; 0.05). Qualitative FTIR suggested greater preservation of phosphate-related bands in FCPS and NM; however, no differences were observed in the phosphate-to-amide I ratio (<i>P</i> = 0.72).</p> Conclusions <p>Within the limitations of this in vitro study, dentifrices influenced the durability of in-office desensitizing treatment under erosive–abrasive conditions. FCPS demonstrated greater stability over time, while FCPS and NovaMin^® were associated with increased tubule occlusion. These findings suggest that bioactive dentifrices may help maintain desensitizing effects; however, clinical studies are required to confirm their long-term effectiveness.</p>