Aim: The aim of the current research was to evaluate the effectiveness of three desensitizing agents on the occlusion of dentinal tubules. Materials and Methods: Eighty human maxillary and mandibular permanent premolar teeth that were subjected to extraction in the course of orthodontic treatment were utilized in this study. The teeth were subjected to sectioning by employing a double-sided diamond disk. A block was procured from every even cervical dentinal surface with dimensions of 4 mm × 4 mm × 3 mm (L × B × H) by sectioning the tooth transversely. Further, 35% phosphoric acid was utilized for 30 s to cause dentinal etching, thereby exposing the dentinal tubules. The blocks of dentin were assigned at random to one of the following four groups (n = 20): group I: control, group II: bioactive glass containing desensitizing agent, group III: 1% nano-hydroxyapatite containing desensitizing agent, group IV: tri-calcium phosphate-containing desensitizing agent. The exterior of the specimens was scanned and evaluated using a scanning electron microscope. Results: 1% nano-hydroxyapatite-containing desensitizing agent (2.04 ± 0.08) exhibited higher mean dentinal tubular blockage, followed by the bioactive class-containing desensitizing substance (2.26 ± 0.19), tri-calcium phosphate desensitizing agent (2.84 ± 0.10), and the control group (4.18 ± 0.12) in that order. Amid the experimental groups employing the various desensitizing substances, there were significant differences statistically with P < 0.001. Conclusion: The current research concluded that each of the three desensitizing agents was efficient in dentinal tubular occlusion regardless of their different chemical constitution. However, it was noted that 1% nano-hydroxyapatite-containing desensitizing agent was marginally superior to the bioactive glass- and tri-calcium phosphate-containing desensitizing agents.