Two different composite scaffolds, solid-freeform-fabricated PCL/β-TCP supplemented with and without collagen nanofibers are fabricated. These scaffolds are evaluated whether a combination of collagen nanofibers with PCL/β-TCP can promote osteogenesis in a mastoid obliteration. To assess the effects of the cellular activities of osteoblast-like-cells (MG63), SEM images and MTT assays are conducted. Experimental mastoid obliteration is performed using guinea pigs that are divided group A (PCL/β-TCP/collagen-nanofiber scaffold) and group B (PCL/β-TCP scaffold). The results reveal that PCL/β-TCP/collagen scaffold provide much broader cell attachment sites than PCL/β-TCP scaffold. The µ-CT and fluorescent microscopy results reveal that the acceleration of early new bone formation within the pores and scaffold itself at week 4 post-operation is more effective in group A. In addition, based on the results of the histological and µ-CT at 12 weeks post-surgery, the effective regeneration of bone in the PCL/β-TCP/collagen scaffold is appeared.