Background: This study was performed to examine the effect of Whey extract and nano-hydroxyapatite powder mixed with olive in different times of application on the microhardness and micromorphology of white spot lesion. Materials and Methods: A total of 16 premolars were selected. The samples were sectioned mesiodistally into two halves to obtain 32 specimens, then divided into two groups according to materials used; A1: Whey extract, A2: Nano-hydroxyapatite paste. The two groups were subdivided into two subgroups according to the time of assessment; B1: after 10 minutes and B2: after 8 days. All the samples were treated by demineralizing solution for 72 hours, and then the tested materials were applied. Microhardness test was performed three times for each sample, at baseline, after demineralization, and after treatment for each period of time (after 10 minutes and after 8 days). Two representative samples from each group (one representing 10 minutes and the other representing 8 days) and two samples of sound and one for demineralized enamel were selected for SEM. Results: Microhardness results revealed that the two materials show a significant increase in microhardness with no significant difference between them. SEM demonstrated improvement in demineralized enamel surface defects in the two test groups. Conclusion: Whey extract and nano-hydroxyapatite powder mixed with olive oil are considered an effective therapy to re-mineralize the white spot lesion of the enamel surface regardless of the time of application (10 minutes or 8 days).