Approximately 10% of all fractures result in delayed healing or non-unions. Bone healing can be improved by the application of osteoconductive and osteoinductive biomaterials. Microcalcite (MCA) as a naturally available calcium carbonate-based biomaterial derived from marble may have the potential to improve bone healing. Herein, we studied for the first time, if MCA in combination with platelet-rich plasma (PRP) can be used as a bone graft material for bone healing in vivo. For this purpose, osteotomies were induced in CD-1 mice (n = 60). Animals received into the osteotomy gap either MCA-loaded PRP (MCA + PRP; n = 20), PRP alone (PRP; n = 20) or no application (NONE; n = 20). Bone healing was evaluated at two and five weeks after osteotomy by micro-computed tomography (μCT), histomorphometric, immunohistochemical and Western Blot analyses. μCT of MCA + PRP femurs revealed more bone volume and an increased polar moment of inertia, indicating a higher biomechanical stability when compared to PRP and NONE femurs. Histomorphometry revealed an increased total callus area after two weeks and a reduced callus tissue area after five weeks in MCA + PRP and PRP animals compared to NONE animals, indicating an accelerated process of bone healing and remodeling over the study period. Moreover, histomorphometric analyses demonstrated an increased fraction of osseous tissue within the callus in MCA + PRP femurs when compared to PRP and NONE femurs. Immunohistochemical analyses showed increased numbers of Ki67+ cells in callus tissue of MCA + PRP femurs. Of interest, Western Blotting revealed a significantly reduced expression of BMP-4 in MCA + PRP animals, while the expression of BMP-2 did not reveal any significant differences between the groups. This indicates a modified balance between angiogenesis and osteogenesis due to MCA. In conclusion, the application of MCA with PRP improved bone healing in a murine osteotomy model and, thus, might be a promising novel bone graft material which may be of interest for clinical fracture treatment.