Objectives: Dentine hypersensitivity (DH) is a common concern in dentistry that has the potential to restrict daily activities and harm a person’s quality of life. In this study, the remineralization characteristics of nano-hydroxyapatite (nHAp) extracted from waste eggshells and fish scales were comparatively assessed. Materials and methods: The extraction methods used to obtain nHAp from both fish scales and eggshells are also described. The effect of the extraction process and bio-waste source on the physicochemical characteristics of the nHAp such as Ca/P ratio, functional groups, crystallinity and phase change, and surface morphology are presented in the study. The remineralization properties were evaluated using dentine models ( n = 15). A field scanning electron microscope was used to evaluate the effectiveness of the dentine tubules occlusion. The percentage occluded area for all the specimens was evaluated statistically using a one-way analysis of variance (α = 0.05). Results: The results showed that there were variations in the physicochemical characteristics of the nHAp extracted, including the crystallinity, particle size, and surface morphology, and buffering effects against citric acids. The EnHAp extracted from eggshells had higher crystallinity, superior buffering effects, and smaller particle size compared to the nHAp extracted from fish scales, making it a more favourable material for remineralization of teeth. The statistical evidence showed that there were statistically significant differences in the dentine occluding properties measured in the nHAp ( p < 0.001). The highest mean % occluded area was measured with the EnHAp group. Conclusions: The findings of this study provide insights into the use of bio-waste materials for the development of sustainable and effective products for oral health care.