Background and objective The effectiveness of various remineralizing agents, including fluoride varnish, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), and chicken eggshell powder (CESP), in improving enamel surface microhardness (SMH) is of clinical interest. This in vitro study aimed to evaluate and compare SMH changes in the enamel after treatment with these agents. Materials and methods An in vitro study was conducted in which 40 human premolars extracted for orthodontic reasons were sectioned into four enamel sections, which were randomly allocated into four groups: group A (fluoride varnish), group B (CPP-ACP cream), group C (CESP), and group D (control). Group A was treated with a thin layer of fluoride varnish (MI Varnish®, GC Corp., Tokyo, Japan), which was left to absorb for 20 s before air drying. Group B specimens were treated with CPP-ACP cream (GC Tooth Mousse®
GC Corp.) for at least three minutes. Group C received CESP application to the enamel surface, which was allowed to absorb for five minutes. Group D (the control group) did not receive any surface treatment. Artificial carious lesions were induced using a demineralization solution and pH cycling for five days. The treated samples were stored in artificial saliva for 21 days at 37 °C. The SMH was assessed using a Vickers microhardness tester at baseline, post-demineralization, and post-treatment. Statistical analyses included paired t-test, one-way analysis of variance (ANOVA), and Tukey's post-hoc test. Results After demineralization, SMH significantly decreased in all groups (p<
0.05). After treatment with various remineralizing agents, groups A, B, and C demonstrated significant improvement in SMH compared to the control group (p<
0.05). Group C exhibited the greatest improvement in SMH, followed by groups A and B. The difference in the SMH between the treatment groups was statistically significant (p<
0.05). Conclusions All three remineralizing agents effectively increased SMH, with CESP showing superior results. Fluoride varnish and CPP-ACP also demonstrated substantial remineralization potential. Further in vivo studies are required to validate these findings in a clinical setting.