Introduction: Investigations have demonstrated that fluoride is an essential element in preventive dentistry. However, there are still controversies about the preventive effects of various kinds of laser. The aim of this study was to examine the effect of diode laser irradiation (810 nm) with or without fluoride therapy in the prevention of deciduous enamel demineralization. Methods: Sixty deciduous molar crowns were randomly assigned to 6 groups: C: received no treatment; F: fluoride varnish application; 2L: 2 times diode laser irradiation; 4L: 4 times diode laser irradiation; F2L: 2 times laser irradiation over fluoride varnish; F4L: 4 times laser irradiation over fluoride varnish. Teeth in all groups were subjected to a pH-cycling process to produce artificial caries-like lesions. Results: The analysis of variance (ANOVA) of microhardness values indicated a significant great effect for laser, fluoride, and the interaction of laser-fluoride on reducing the final microhardness value (P < 0.001). However, the 2L group was an exception. Despite the 4L group, it did not show a significant prevention of enamel microhardness loss (P = 0.125). These 2 groups exhibited different effects in the absence of fluoride (P 2L-4L = 0.05) while in the presence of the fluoride varnish, no statistically significant difference was observed between them (P F2L-F4L = 0.257). Moreover, no statistically significant difference was observed between the laser-fluoride combination group and the fluoride group (P F2L-F = 0.133, P F4L-F = 0.926). Conclusion: Our results suggest that fluoride varnish, diode laser, and their combination decrease the loss of the enamel microhardness value and potentially prevent deciduous enamel demineralization. However, the combination of laser and fluoride was not more effective than fluoride.