Fluoride (F) has been a useful instrument in caries prevention. However, only limited data exist on the effect of its long-term use on dentin mineralization patterns and microhardness. The objective of this study was to evaluate the influence of tooth F concentration ([F]) and dental fluorosis (DF) severity on dentin microhardness and mineralization. We collected 137 teeth in Montreal and Toronto, Canada, and Fortaleza, Brazil, where optimum or suboptimum levels of water F were 0.2 ppm, 1 ppm, and 0.7 ppm, respectively. Teeth were analyzed for DF severity, dentin [F], enamel [F], dentin microhardness, and dentin mineralization. Dentin [F] correlated with DF severity; enamel [F] correlated with dentin microhardness and dentin mineralization; DF severity correlated with dentin microhardness. Genetic factors (e.g., DF severity) and environmental factors (e.g., tooth [F]) influenced the mechanical properties (microhardness) of the teeth, while only the environmental factors influenced their material properties (e.g., mineralization). Fortaleza teeth were harder and less mineralized and presented higher dentin [F] values. Montreal teeth presented lower levels of DF when compared with both Toronto and Fortaleza teeth.