This study aims to assess the effect of low-level laser therapy (LLLT) irradiation and Bio-Oss graft material on the osteogenesis process in the rabbit calvarium defects. Twelve white male New Zealand rabbits were included in this study. Four 8-mm diameter identical defects were prepared on each rabbit's calvarium. One site was left as an untreated control (C), the second site was filled with Bio-Oss (B), the third site was treated with laser irradiation (L), and the fourth site treated with Bio-Oss and laser irradiation (B + L). In the laser group, a diode laser (wavelength 810 nm, output power 300 mW, irradiation mode CW, energy density 4 J/cm2) was applied immediately after surgery and then one other day for the next 20 days. After 4 and 8 weeks, the animals were sacrificed and histological and histomorphometric examinations were performed and the data were subjected to Friedman and repeated measurements ANOVA tests. Significant differences were not found regarding inflammation severity, foreign body reactions, and vitality of newly formed bone on 4th and 8th week after operation. The mean amount of new bone was 15.83 and 18.5% in the controls on the 4th and 8th week; 27.66 and 25.16% in the laser-irradiated group; 35.0 and 41.83% in Bio-Oss and 41.83 and 47.0% in the laser + Bio-Oss treated specimens with significant statistical differences (p <0.05). Application of LLLT in combination with Bio-Oss® can promote bone healing. Therefore, LLLT may be clinically beneficial in promoting bone formation in skeletal defects.