Background: Dentinal hypersensitivity is a prevalent dental condition characterized by sharp pain in response to thermal, tactile, or chemical stimuli due to exposed dentinal tubules. Effective management aims to occlude these tubules to reduce sensitivity. Combining desensitizing dentifrices with diode laser therapy has emerged as a potential strategy to enhance tubule occlusion. Methods: This in vitro study evaluated the synergistic effects of two dentifrices—Calcium Sodium Phosphosilicate (Novamin) and Fluoro Calcium Phospho Silicate (Biomin)—in combination with diode laser therapy on dentinal tubule occlusion. A total of 120 dentin specimens from extracted sound premolars were divided into six groups: Group 1 (Biomin), Group 2 (Novamin), Group 3 (Biomin + Diode Laser), Group 4 (Novamin + Diode Laser), Group 5 (Diode Laser alone), and Group 6 (Distilled Water Control). Treatments were applied using a battery-powered toothbrush, followed by laser irradiation where applicable. Scanning Electron Microscopy (SEM) was utilized to assess tubule occlusion, and data were analyzed using one-way ANOVA. Results: Group 3 (Biomin + Diode Laser) exhibited the highest mean percentage of completely occluded tubules (79.27%), significantly outperforming other groups. Group 1 (Biomin alone) followed by Group 4 (Novamin + Diode Laser) also demonstrated substantial occlusion. The control group showed minimal occlusion (2.68%). Partial occlusion was highest in Group 5 (Diode Laser alone), while non-occluded tubules were significantly reduced in all experimental groups compared to control. Conclusion: The combination of Fluoro Calcium Phospho Silicate (Biomin) with diode laser therapy significantly enhances dentinal tubule occlusion compared to individual treatments, suggesting a potential effective treatment modality for patients with dentinal hypersensitivity.