Osteochondral lesions, which affect both the cartilage and the bone, present significant challenges in treatment due to the complex mechanical and biochemical properties of these tissues. A crucial consideration in developing tissue replacements for these lesions is the simultaneous regeneration of cartilage and calcified cartilage, which forms the transition zone to bone. Our current study aims to fabricate a bilayer polymeric hydrogel designed not only to support cartilage regeneration but also to serve as an interface between cartilage and bone. The bilayer hydrogel was created by combining oxidized bacterial nanocellulose, gelatin, and alginate in one layer, while the other layer consisted of the same three biopolymers and hydroxyapatite. The bacterial nanocellulose was effectively oxidized (20%) with sodium periodate and then mineralized with calcium and phosphorus (Ca/P ratio = 0.97), as confirmed by EDX analysis. Remarkably, both layers of the biphasic hydrogel demonstrated cytocompatibility with chondrocytes. Moreover, the addition of hydroxyapatite significantly improved the mechanical strength from 72 kPa (OBC/Gel/Alg) to 90 kPa (MOBC/Gel/Alg). This bilayer hydrogel holds promise for promoting bone-cartilage integration and has the potential to contribute to the healing of osteochondral defects, offering new possibilities in the field of orthopedic tissue engineering and regenerative medicine.