Developing multifunctional systems for the biomimetic remineralization of human enamel is a challenging task, since hydroxyapatite (HAP) rod structures of tooth enamel are difficult to replicate artificially. The paper presents the first report on the simultaneous use of chitosan (CS) and agarose (A) in a biopolymer-based hydrogel for the biomimetic remineralization of an acid-etched native enamel surface during 4–10-day immersion in artificial saliva with or without (control group) fluoride. Scanning electron microscopy coupled with energy-dispersive X-ray spectrometry, Fourier transform infrared and Raman spectroscopies, X-ray diffraction, and microhardness tests were applied to investigate the properties of the acid-etched and remineralized dental enamel layers under A and CS-A hydrogels. The results show that all biomimetic epitaxial reconstructed layers consist mostly of a similar hierarchical HAP structure to the native enamel from nano- to microscale. An analogous Ca/P ratio (1.64) to natural tooth enamel and microhardness recovery of 77.4% of the enamel-like layer are obtained by a 7-day remineralization process in artificial saliva under CS-A hydrogels. The CS component reduced carbonation and moderated the formation of HAP nanorods in addition to providing an extracellular matrix to support growing enamel-like structures. Such activity lacked in samples exposed to A-hydrogel only. These data suggest the potential of the CS-A hydrogel in guiding the formation of hard tissues as dental enamel.