Vanadium is a trace element in the human body, and vanadium compounds have a promising future in biological and medical applications due to their various biological activities and low toxicity. Herein, a novel pure vanadium dioxide (VO) nanofilm was deposited on a substrate of biomedical titanium by magnetron sputtering. The antibacterial effect of VO against the methicillin-resistant Staphylococcus aureus (MRSA) was validated in vitro and in vivo. Moreover, the biocompatibility of VO and its osteogenic effects were systematically illustrated. A possible osteogenic mechanism involving the amelioration of highly reactive oxygen species (ROS) levels were investigated. According to the results of our present and previous studies, the simultaneous antibacterial and osteogenic effects of VO are attributed to its differential regulation of ROS levels in rat bone marrow mesenchymal stem cells (rBMSCs) and bacteria. This study is the first to report the simultaneous effects of VO on bactericidal and osteogenic activities through its differential modification of ROS activity in eukaryotic (rBMSCs) and prokaryotic (MRSA) cells. The findings in this work may yield a deeper understanding of the biological activities of vanadium compounds while also paving the way for the further investigation and application of VO in biological and medical materials.