Bone disorders and skeletal defects represent a significant clinical challenge, often requiring transplantation techniques limited by donor site morbidity and insufficient regenerative potential. Tissue engineering and regenerative medicine (TERM) strategies using 3D bioprinting have emerged as promising alternatives, but their efficacy is limited by the difficulty of directing stem cell differentiation in a controlled and reproducible manner. To address this limitation, we are proposing 3D bone printing via ultrasound-mediated osteogenic differentiation of stem cells (referred to here as '3DBonUS'). This biofabrication approach integrates low-intensity pulsed ultrasound (LIPUS) with a microfluidic-assisted 3D bioprinting system. This unprecedented approach enables biophysical stimulation of human bone marrow stromal cells (HBMSCs) during the fabrication of scaffolds, promoting osteogenic differentiation without the need for extensive post-fabrication treatments. In addition, the incorporation of microbubbles (MBs) enhanced the effects of LIPUS by amplifying mechanical signals at the cellular level. Our results revealed that the 3DBonUS system significantly upregulated key osteogenic markers (RUNX-2, ALP, COL1A1, BMP-2, OCN and OPN) as confirmed by immunofluorescence and RT-qPCR analysis. Moreover, the LIPUS-treated constructs showed a significant (p<0.05) increase in alkaline phosphatase (ALP) activity and calcium deposition, indicating enhanced mineralisation. The biofabricated constructs maintained high cell viability while exhibiting improved osteogenic differentiation, surpassing traditional 3D bioprinting approaches in both efficiency and efficacy. The 3DBonUS strategy represents a new modality in skeletal TERM, combining biofabrication with targeted mechanical stimulation, with potential for scalability of scaffold manufacturing and clinical application. Future studies will aim to validate the functional skeletal scaffolds in vivo to assess their regenerative potential, with the goal of advancing patient-specific bone implants with enhanced osteogenic properties.