The objective of this in vitro study was to analyze and compare the biomimetic remineralizing efficacy of the self-assembling peptide (P11-4) with agents containing casein phoshopeptide-amorhous calcium phosphate fluoride (CPP-ACFP) and sodium fluoride (NaF) on artificial caries lesions using DIAGNOdent and micro-computed tomography (mu CT). Artificial enamel lesions were prepared on extracted impacted sound mandibular third molars. The samples were randomly allocated to four groups (n = 8): Group 1, P11-4 (Curodont Repair, Credentis AG, Switzerland); Group 2, CPP-ACFP (MI Varnish, GCCo., Japan); Group3, NaF (Duraphat Varnish, Colgate, Colgate-Palmolive, NY, USA); Group 4, artificial saliva (control). The agents were applied to demineralized surfaces according to manufacturers' instructions; all specimens were stored in artificial saliva for 1 month. Demineralization and remineralization on enamel surfaces were analyzed and quantified by DIAGNOdent (KaVo, Germany) and mu CT (SkyScan1174, Belgium) for lesion depth/area/volume/mineral density (MD). The remineralization efficacy of the agents was evaluated by DIAGNOdent on 1st, 7th, 30th days and by mu CT on 30th day. Data were statistically analyzed by ANOVA, Kruskal-Wallis, T test, and Wilxocon tests. The highest remineralization efficacy findings in all periods were determined in Group 1, followed by Groups 2, 3, and 4. The remineralization findings for fluorescence, MD, lesion depth in Group 1 were found significantly higher (p < 0.01) than Group 3; and no significant differences (p > 0.05) were found between Groups 1-2 and Groups 2-3. The area and volume change values in Groups 1, 2, and 3 have shown no significancy (p > 0.05). A significant correlation (p < 0.01) was found between mu CT and DIAGNOdent methods. The data of this study have demonstrated that P11-4 has showed the best remineralization efficacy, followed by CPP-ACFP and NaF. It is concluded that self-assembling peptide-based remineralization agent can be used successfully for biomimetic remineralization of enamel subsurface lesions.