The aim of this study was to evaluate the effect of 3D 2% w/v chitosan scaffolds on new bone formation in a rabbit calvarial defect. Two bi-cortical skull defects were prepared in 16 male rabbits and divided into two groups: autogenous (positive controls) and chitosan scaffolds (experimental groups). The animals were sacrificed at 2, 4, 8, and 12 weeks. New bone formation was evaluated by micro-CT and histomorphometric analysis. The positive control group demonstrated significantly greater bone formation. The experimental groups showed viable lamellar bone with osteoblasts forming bone from the defect margin. Histomorphometric analysis showed an increased percentage of new bone formation over time, and a micro-CT analysis also showed an increased percentage of bone volume over time. Although the new bone formation was not as good as the positive control groups, our in-house fabricated chitosan scaffolds presented good biocompatibility and osteoconductive properties in a rabbit calvarial defect.