Background: Dentin hypersensitivity is primarily caused due to patent or exposed dentinal tubules. Nonfluoridated?desensitizing agents deposit hydroxycarbonate apatite (HCA), within the dentinal tubules, thereby relieving hypersensitivity. Fluoride?containing bioactive glass?based agents form fluorapatite which is less soluble when compared to hydroxyapatite and HCA. Materials and Methods: In this in vitro study forty dentin specimens obtained from extracted human premolars were divided randomly into four groups (n = 10): Group 1 – fluoridated bioactive glass (FBaG); Group 2 – bioactive glass (BaG); Group 3 – arginine calcium carbonate; Group 4 – saline. 37.5% phosphoric acid was used to ensure patent dentinal tubules. Test agents from each group were applied using a rubber cup. Half the treated samples were then subjected to 6% citric acid treatment. The degree of occlusion was evaluated using the scanning electron microscope, and the microscopic images were scored before and after the citric acid challenge by two blinded endodontists. Statistical analysis was performed using SPSS, one?way ANOVA, and post hoc Tukey test (P = 0.05). Results: Group 1 demonstrated better tubule occlusion in comparison with Group 2 and a statistically significant difference when compared to Group 3 (P = 0.001). Following acid challenge, Group 2 showed significantly more occluded tubules when compared with Group 3 (P = 0.001) and comparable difference with Group 1. Conclusion: All desensitizing agents showed satisfactory dentinal tubule occlusion. While fluoridated bioactive glass demonstrated better occlusion immediately after application, Bioactive glass showed better resistance to acid treatment.