This study presents the development of biocompatible and 3D printable hydrogels composed of quaternized chitosan (DQC), non-isocyanate polyurethane (NIPU-Cys), and TEMPO-oxidized cellulose nanofibrils (CNF) for wound-healing applications. The hydrogels were synthesized by combining DQC with NIPU-Cys and CNF to enhance mechanical properties, flexibility, and biocompatibility. The hydrogels were evaluated for their swelling capacity, mechanical properties, and in vitro biocompatibility. The results showed that the hydrogels exhibited good swelling ratios, porous architecture, and favorable mechanical properties. The incorporation of DQC enhanced in vitro cell proliferation on the scaffolds without negatively impacting cell viability. The hydrogels were found to be suitable for 3D printing via direct ink writing (DIW) and demonstrated good biocompatibility with fibroblast cells. The study also highlights the potential of these hydrogels as biocompatible materials for various biomedical applications. The results indicate that the slightly cytotoxic nature of quaternary chitosan is mitigated when incorporated into an NIPU-CNF matrix. The combination of DQC in a NIPU-Cys-CNF matrix emerges as a promising composition for crafting 3D-printable, biocompatible, and porous hydrogels with excellent swelling properties. These materials hold significant potential as constituents for creating biocompatible materials for various biomedical applications.This study presents the development of biocompatible and 3D printable hydrogels composed of quaternized chitosan (DQC), non-isocyanate polyurethane (NIPU-Cys), and TEMPO-oxidized cellulose nanofibrils (CNF) for wound-healing applications. The hydrogels were synthesized by combining DQC with NIPU-Cys and CNF to enhance mechanical properties, flexibility, and biocompatibility. The hydrogels were evaluated for their swelling capacity, mechanical properties, and in vitro biocompatibility. The results showed that the hydrogels exhibited good swelling ratios, porous architecture, and favorable mechanical properties. The incorporation of DQC enhanced in vitro cell proliferation on the scaffolds without negatively impacting cell viability. The hydrogels were found to be suitable for 3D printing via direct ink writing (DIW) and demonstrated good biocompatibility with fibroblast cells. The study also highlights the potential of these hydrogels as biocompatible materials for various biomedical applications. The results indicate that the slightly cytotoxic nature of quaternary chitosan is mitigated when incorporated into an NIPU-CNF matrix. The combination of DQC in a NIPU-Cys-CNF matrix emerges as a promising composition for crafting 3D-printable, biocompatible, and porous hydrogels with excellent swelling properties. These materials hold significant potential as constituents for creating biocompatible materials for various biomedical applications.