Purpose: To evaluate the effects of stomach and duodenal fluid on enamel surfaces, simulating the action of refluxed liquid in patients with duodenogastric reflux. Methods and Materials: Forty bovine incisors were used to obtain enamel fragments. Only half of the enamel surface was exposed to erosive challenges; the samples were then randomly divided into the following four groups (n = 10): G1: HCl; G2: HCl + pepsin; G3: HCl + ox bile + NaHCO3; and G4: HCl + pancreatin + NaHCO3. The specimens were placed in 37°C solutions, six times per day, for 20 s, over a period of 5 days and then analysed for morphology, surface roughness and the step formed on the dental enamel using confocal laser microscopy. The data were analysed using the Kruskal-Wallis and Dunn's test (p < 0.05). Results: Both analyses revealed a higher step and surface roughness for the G3 group (5.6 μm ± 1.69, 2.2 μm ± 1.61), which were statistically significant compared with the G1 and G2 groups (3.9 μm ± 1.5 μm; 1.0 μm ± 0.18; 3.7 μm ± 1.45; and 0.9 μm ± 0.12) (p < 0.05); only the step in the G4 group (4.9 μm ± 1.8 μm) was similar to that of the G3 group (p > 0.05). Morphological analysis showed greater structural loss in the G3 and G4 groups. Conclusions: Bile and pancreatin, in combination with hydrochloric acid, may promote a greater loss of structure, increased surface roughness and loss of enamel prismatic anatomy.