Background: Fluoride (F) is an effective anticaries agent and can be delivered through various mediums at different concentrations. The ability to increase the enamel resistance to acid by decreasing solubility through the incorporation of F into the enamel apatite structure is the primary function of these agents. The efficacy of topical F can be determined by measuring the amount of F incorporated in and on human enamel. Aim: To compare the F uptake into and onto the enamel surface of two different F varnishes at different temperatures. Materials and methods: In this study, 96 teeth were divided equally and randomly (n = 48) into two experimental groups (group I and group II). Each group was further divided into four equal subgroups (n = 12); depending on the temperature (25°, 37°, 50°, and 60°C) they were subjected to Fluor-Protector® 0.7% and Embrace® 5% F varnishes were allocated to experimental groups I and II, respectively, and every sample was individually treated with the assigned F varnish. After the varnish application, two specimens from each subgroup, the group I and group II (n = 16), were mounted for hard tissue microtome sectioning for scanning electron microscope (SEM) analysis. The remaining 80 teeth underwent potassium hydroxide (KOH) soluble and KOH-insoluble F estimation. Results: Group I and group II, both, showed maximum F uptake of 2817.07 ppm and 1626.8 ppm at 37°C temperature and the lowest of 1168.9 ppm and 1068.93 ppm at 50°C, respectively. The intergroup comparison was carried out using an unpaired t-test and the intragroup comparison was done using one-way analysis of variance (ANOVA) univariate analysis. Post hoc Tukey test was performed for pairwise comparison between different temperature groups. In group I (Fluor-Protector®), the difference in F uptake was statistically significant when the temperature was increased from 25° to 37° C (mean difference = −9.90, p = 0.016). In group II (Embrace®), a statistically significant difference in F uptake was observed when the temperature was increased from 25° to 50°C (mean difference = 10.00, p = 0.003) and 25° to 60°C (mean difference = 13.38, p = 0.001), respectively. Conclusion: Fluor-Protector® varnish proved to have better F uptake in comparison to Embrace® varnish on human enamel. Topical F varnishes were most effective at 37°C, which is close to the standard human body temperature. Thus, the application of warm F varnish ensures more uptake of F in and onto the enamel surface for greater protection against dental caries.