Background Development of white spot lesions around orthodontic fixed orthodontic appliances is a common finding, especially in patients with poor oral hygiene. One of the conservative interventions for regression of these lesions is using chemical solutions. The current study aimed to compare the effectiveness of fluoride and amorphous calcium phosphate (ACP) on microhardness improvement of affected enamel. Material and Methods Forty-five intact human incisor teeth were selected and randomly divided into 3 groups of 15. Fluoride group, ACP group and artificial saliva group (control group). Inducing of white spot lesion was done by PH-cycling model. Samples of the first and second group were submerged into 0.05% fluoride and 0.05% ACP solutions respectively for one minute a day. The rest of the time, all specimens were put in artificial saliva, which was incubated in 37 °c temperature. Microhardness of specimens was assessed by Vickers microhardness test in three stages: 1: Baseline microhardness assessment that was done before induction of white spot lesion, 2: Secondary microhardness assessment that was done after induction, 3: Final microhardness assessment that was done after chemical treatment. The SPSS 11.5 software was used for statistical analysis and p < 0.05 was considered as significant. Results Microhardness of specimens in the fluoride and ACP groups had significantly improved after the treatment (between secondary assessment and final assessment). In the control group, no significant improvements were observed. In final assessment, there were significant differences between the ACP and control groups, but no significant differences were found neither between the fluoride and ACP, nor the Fluoride and control groups. Conclusions According to the current study, both 0.05% ACP and 0.05% fluoride solutions enhanced enamel micro-hardness in treatment of white spot lesion. Key words: Microhardness, amorphous calcium phosphate, fluoride, white spot lesion.