Despite fluoride (F) use in caries prevention, not much is known about its effects on tooth quality. This study evaluated the effect of tooth F concentration ([F]) on selected dentin structural and mechanical properties. Third molars (n = 136) from Toronto, which has 1 part per million (p.p.m.) water [F], Montreal (0.2 p.p.m. water [F]), and Fortaleza (Brazil) (0.7 p.p.m. water [F]), were analyzed for [F], dental fluorosis (DF) severity, ultrasound velocity, and dentin tubule size and density. The enamel [F] was found to vary between 32 and 940 p.p.m., the dentin [F] was found to vary between 110 and 860 p.p.m., while the DF severity varied between TF0 and TF4. The enamel [F] showed no correlation with dentin [F], DF severity, ultrasound velocity, dentin tubule size or density. The dentin [F] correlated with DF severity, dentin tubule size, and ultrasound velocity. DF severity showed a correlation with dentin [F] and ultrasound velocity. It was concluded that dentin [F] is an indicator of dentin structural properties (dentin tubule size and ultrasound velocity), while DF severity is an indicator of dentin mechanical properties (ultrasound velocity).