This study presents the development and characterization of biocompatible and 3D printable hydrogels for wound-healing applications. The hydrogels were synthesized by combining antibacterial double-quaternized chitosan (DQC) with cystamine-based non-isocyanate polyurethane (NIPU-Cys) and cellulose nanofibrils (CNF). The impact of DQC on degradation, swelling capacity, rheological behavior, printability, and cell biocompatibility was investigated. The optimal concentration of DQC was determined to ensure biocompatibility. The resulting hydrogels were suitable for 3D printing via direct ink writing (DIW) technology, producing porous structures with valuable attributes for wound healing. The hydrogels exhibited no acute harmful effects on fibroblast cells, making them suitable as biocompatible barrier layers. The study highlights the potential of these hydrogels in biomedical applications, particularly in wound management.This study presents the development and characterization of biocompatible and 3D printable hydrogels for wound-healing applications. The hydrogels were synthesized by combining antibacterial double-quaternized chitosan (DQC) with cystamine-based non-isocyanate polyurethane (NIPU-Cys) and cellulose nanofibrils (CNF). The impact of DQC on degradation, swelling capacity, rheological behavior, printability, and cell biocompatibility was investigated. The optimal concentration of DQC was determined to ensure biocompatibility. The resulting hydrogels were suitable for 3D printing via direct ink writing (DIW) technology, producing porous structures with valuable attributes for wound healing. The hydrogels exhibited no acute harmful effects on fibroblast cells, making them suitable as biocompatible barrier layers. The study highlights the potential of these hydrogels in biomedical applications, particularly in wound management.