The high costs and extensive time needed for the treatment of critical-sized bone defects are still major clinical concerns in orthopedic surgery; therefore, researchers continue to look for more cost and time-effective methods. This study aims to investigate the effects of a decellularized omentum scaffold with a combination of platelet-rich plasma (PRP) and mesenchymal stem cells on the healing of critical-sized bone defects. Wistar albino rats (n = 30) were investigated in five groups. Critical-sized bone defects were formed on bilateral radius shafts. No scaffold, decellularized omentum, omentum with PRP and omentum + mesenchymal stem cells was used in group 1 (control group), 2, 3 and 4, respectively. In addition, omentum with a combination of mesenchymal stem cells +PRP was used in group 5. After 6 weeks, both radiological and histological healing were evaluated comparatively among the groups. After the use of a decellularized omentum scaffold, vitality of new cells was maintained, and new bone formation occurred. When compared to the control group, radiological healing was significantly better (p = 0.047) in the omentum and omentum + PRP-treated groups. Furthermore, histological healing was better in the omentum and omentum + PRP-treated groups than the control group (p = 0.001). The use of a decellularized omentum scaffold is suitable in the healing of critical bone defects.