Iontophoresis is generally used to maximize the therapeutic action of drugs in medicine. This technique can be used to improve the remineralization effect of topical fluoride applications in dentistry. The aim of this study was to compare the remineralization effect of fluoride iontophoresis (FI) with the conventional fluoride application (CFA) method in vitro. Sixty bovine enamel specimens were divided into three groups: no fluoride treatment, CFA and FI. Fluoride was applied to the demineralized specimens for 4 min in each experimental group. The types of fluoride system used for application were 1.23% acidulated phosphate fluoride gel (12 300 p.p.m. F, pH 3.5) and 2% sodium fluoride solution (9050 p.p.m. F, pH 7) in the experimental groups. All the specimens were then placed in a remineralizing solution for 24 h. This cycle was repeated five times. An iontophoresis device (0.4 mA, 12 V) was used in the FI groups. The efficacy of this technique was evaluated by measuring changes in the surface microhardness and lesion depth of the specimens using confocal laser scanning microscope (CLSM). Data were analysed using anova and Tukey's post hoc test (P < 0.05). Although the FI groups showed higher DeltaVHN than the CFA groups, there were no significant differences between these fluoride application methods (P > 0.05). When the lesion depth was measured using CLSM imaging, there was also no significant difference between the FI and CFA groups (P > 0.05). In conclusion, FI was not significantly superior to CFA in terms of the remineralization effect.