Enamel erosion has become a common clinical finding that often impairs dental esthetics and function. In the current study, we aimed to implement the three-dimensional surface texture analysis (3DST) method to explore the protective effect of fluoride on surface texture prior to erosive conditions. For each of the 50 teeth used in this study, the polished buccal enamel surface was divided into three separate areas: the first area was untouched polished enamel, the two other surfaces were immersed in 0.3% citric acid for 30 s. One was treated with high-concentration (19,000 ppm) sodium fluoride (NaF) solution prior to acid attack, and the other had no treatment prior to acid exposure. Enamel surface texture and step height measurements were obtained using a high-resolution disk scanning confocal microscope, and SEM images were also acquired. Surfaces treated with fluoride showed fewer variations in 3-D surface texture parameters than the eroded surface compared to the control group (p = 0.001). This was in accordance with the SEM descriptive images. The findings indicate that pre-fluoridated enamel areas were less affected by the acid and showed similar features to the untouched enamel. Moreover, a protective effect of the fluoride treatment against irreversible enamel damage was noted as the surface loss (step-height) was significantly reduced (p = 0.03). The study showed that 3DST analysis is a valuable methodology for detecting and quantifying subtle differences between the surfaces. When exploring the combination of all surface texture parameters, it was revealed that the pre-fluoridated eroded enamel surfaces showed considerable similarity to the untouched enamel.