Purpose: To evaluate mineral content, microhardness, and microstructure of the enamel like structure using agarose hydrogel system and its stability under further acid attack. Materials & Methods: Twenty-four human 1^st premolars were collected. 3 × 3 mm enamel windows were created in the middle third of the buccal surfaces of all teeth on which initial enamel caries like lesions were created . These were treated with agarose hydrogel system. Twelve specimens were analyzed with EDAX and enamel microhardness was measured throughout the study steps. The enamel microstructure was examined under SEM for the remaining specimens. Results: ANOVA test revealed a highly statistically significant difference (p=0.000) among the mean wt. % values of (Ca), (P) content, (Ca/P) ratio and VHN values recorded in all the study steps. The statistical correlation between the wt. % of Ca & micro hardness values revealed a significant positive relation in all study steps (P≤0.05) except after further acid attack, where no significant relation was found (p =0.07). While the correlation between the (P) wt. % & micro hardness revealed a significant positive relation in sound and untreated enamel (p ≤ 0.05), and no significant relation was found in the treated enamel and that subjected to further acid attack (p ≥ 0.05). The statistical correlation between the Ca/P ratio & micro hardness was a significant positive relation (p ≤ 0.05) in all study steps. SEM findings supported the previous results. Conclusions: The tested agarose hydrogel mineralization system promoted an in vitro biomimetic mineralization and enamel prism like tissue formation on the induced enamel caries like lesions However, it was unstable under further acid attack.