Objective A bone defect rat model was established to investigate the osteogenic effect of local delivery two antibiotics (vancomycin and tobramycin powder) on bone regeneration. Methods Twenty-four Sprague-Dawley (SD) male rats (6 to 8 weeks, 200 to 250 g) were used in this study. All these rats were randomly divided into four groups. Based on dose conversion between rat and human via body surface area, the rat dose of two antibiotics was 88 mu g/g and 176 mu g/g for vancomycin and tobramycin, respectively. Con group (no antibiotic), Van group (vancomycin, 88 mu g/g), Tob group (tobramycin 176 mu g/g), and Van+Tob group (vancomycin 88 mu g/g combined with tobramycin 176 mu g/g). A 5.0-mm full-thickness standardized mandibular bone defect was performed with a drill in each rat and different antibiotic powders were placed over the bone defect space, respectively. All these animals were sacrificed after 12 weeks post-operation. The mandible bones were harvested for further radiographic and histologic analysis. The bone volume/total volume (BV/TV) ratio, bone volume (BV), and bone fractional area (BFA) in the defect area via micro-computed tomography (mu CT scanning) were further analyzed. Then, we performed a histological assessment via hematoxylin and eosin (H&E) and Masson's trichrome staining to analyze bone regeneration and also analyze the number of osteoblasts per filed. Results There were no postoperative deaths, signs of vancomycin-related or tobramycin-related toxicity, or signs of systemic illness in any of the four groups. All wounds healed well, and no complications or surgical site infection were observed in all rats. From the mu CT scans analyses, there was less bone regeneration in the Van group than in the Con group (BV/TV: F = 64.29, R-2 = 0.9602; P = 0.0052; BFA: F = 76.17, R-2 = 0.9662, P = 0.0007; BV: F = 194.4, R-2 = 0.9865, P = 0.0022). However, when the tobramycin and vancomycin were combined, an increase in bone defect re-ossification was found in the Van+Tob group than in the Van group (BV/TV: F = 64.29, R-2 = 0.9602, P = 0.0033; BFA: F = 76.17, R-2 = 0.9662, P = 0.0006; BV: F = 194.4, R-2 = 0.9865, P = 0.0033). Routine H&E and Masson staining supported the finding of mu CT scanning. Quantitative indices confirmed that both the bone regeneration and the number of osteoblasts per filed in the defect area was higher in the Van+Tob group than in the Van group (percentage of bone tissue: F = 145.7, R-2 = 0.9562, P = 0.0008; number of osteoblasts per file; F = 67.3, R-2 = 0.9098, P < 0.0001). There was no significant difference between the Con group and the Van+Tob group on the number of osteoblasts each field (F = 145.7, R-2 = 0.9562, P > 0.9999). Conclusion For bone defect, local application of vancomycin combined with tobramycin was recommended over vancomycin alone. This animal study presents data suggesting that the use of local delivery of vancomycin and tobramycin should be investigated further in clinical studies.