Regenerative endodontic procedures (REPs) which aim to promote root development and pulp tissue regeneration in necrotic immature teeth, have emerged as a promising therapeutic approach. A critical determinant of REP success hinges on effective disinfection of the root canal system, which must eliminate microbial contaminants whilst preserving the microenvironment necessary for dental pulp stem cell tissue regeneration. This study reports on the fabrication of biocompatible 3D printed hydrogel scaffolds designed for root canal disinfection. The scaffolds incorporate benzyldimethyldodecylammonium chloride (BDMDAC) a broad-spectrum quaternary ammonium compound characterised by low cytotoxicity and minimal risk of resistance development. BDMDAC loaded gelatin biomaterial inks were systematically evaluated for rheology properties, mechanical stability and drug release properties. Scaffolds containing 150 µg mL−1 and 250 µg mL−1 BDMDAC exhibited excellent antimicrobial efficacy against 5 bacterial pathogens (including 3 endodontic bacteria-Enterococcus faecalis, Porphyromonas gingivalis, and Streptococcus mutans). Cytocompatibility assays using primary human dental pulp stem cells (HDPSCs) derived from 3 donors confirmed over 70% of cell viability. Furthermore, freeze-dried scaffolds demonstrated excellent shelf-life stability for at least six months. Overall, these findings highlight the potential of 3D printed BDMDAC-loaded 3D printed gelatin scaffolds as an effective and cytocompatible platform for root canal disinfection in REPs.