Background: Dental fluorosis requires aesthetic treatment to improve appearance and etching of enamel surfaces with phosphoric acid is a key step for adhesive restorations. The aim of this study was to evaluate surface roughness and a depth profile in healthy and fluorotic enamel before and after phosphoric acid etching at 15, 30 and 60 seconds. Methods: One hundred and sixty enamel samples from third molars with no fluorosis to severe fluorosis were evaluated by atomic force microscopy. Results: Healthy enamel showed a statistically significant difference (p < 0.05) between mean surface roughness at 15 seconds (180.3 nm), 30 seconds (260.9 nm) and 60 seconds (346.5 nm); depth profiles revealed a significant difference for the 60 second treatment (4240.2 nm). For mild fluorosis, there was a statistically significant difference (p < 0.05) between mean surface roughness for 30 second (307.8 nm) and 60 second (346.6 nm) treatments; differences in depth profiles were statistically significant at 15 seconds (2546.7 nm), 30 seconds (3884.2 nm) and 60 seconds (3612.1 nm). For moderate fluorosis, a statistically significant difference (p < 0.05) was observed for surface roughness for 30 second (324.5 nm) and 60 second (396.6 nm) treatments. Conclusions: Surface roughness and depth profile analyses revealed that the best etching results were obtained at 15 seconds for the no fluorosis and mild fluorosis groups, and at 30 seconds for the moderate fluorosis group. Increasing the etching time for severe fluorosis decreased surface roughness and the depth profile, which suggests less micromechanical enamel retention for adhesive bonding applications.