Mastoidectomy is a surgical procedure for the treatment of chronic otitis media. This study investigated the ability of rat stromal vascular fraction cells (rSVF) in combination with polycaprolactone (PCL) scaffolds and osteogenic differentiation-enhancing blood products to promote the regeneration of mastoid bone defect. Twenty male Sprague Dawley rats were randomly divided according to obliteration materials: (1) control, (2) PCL scaffold only, (3) rSVFs + PCL, (4) rSVFs + PCL + platelet-rich plasma, and (5) rSVFs + PCL + whole plasma (WP). At 7 months after transplantation, the rSVFs + PCL + WP group showed remarkable new bone formation in the mastoid. These results indicate that SVFs, PCL scaffolds, and blood products accelerate bone regeneration for mastoid reconstruction. Autologous SVF cells with PCL scaffolds and autologous blood products are promising composites for mastoid reconstruction which can be easily harvested after mastoidectomy. With this approach, the reconstruction of mastoid bone defects can be performed right after mastoidectomy as a one-step procedure which can offer efficiency in the clinical field.