This paper proposes a potential solution to the current issue of developing advanced, biocompatible biomaterials with integrated therapeutic functionality, which would contribute to improving the treatment of skin defects. This study aimed to develop, characterize and evaluate hydrogels based on type I collagen, pectin, alginate and myrtle essential oil, in order to obtain biomaterials with potential in skin regeneration applications. Hydrogels incorporating alginate, pectin, type I collagen and Myrtus communis essential oil were prepared via a multistep procedure comprising homogenization, crosslinking and lyophilization. The obtained hydrogels were characterized by physicochemical and structural methods, such as FTIR spectroscopy, to identify interactions between components; micro-computed tomography, to evaluate internal morphology and porosity; antibacterial tests, for evaluating the ability of the hydrogel to prevent infections at the application site; and in vitro cellular tests, such as the XTT test or cytotoxicity tests, such as LDH, essential for evaluating the biocompatibility of the hydrogel. The highest viability value was recorded for sample J4 (99.53 ± 11.88%), indicating an exceptional compatibility with the cells used, almost identical to that of the untreated control. The samples showed encouraging results, supporting their potential for applications in wound treatment and skin regeneration.