Recently, there has been increased interest in electrospun-titanium dioxide nanofibers (TiO2 NFs) as antibacterial agents owing to their advantages, such as simple and cost-effective fabrication processes, and high surface areas. However, the photocatalytic effects of TiO2 NFs are relatively low because of their low-ordered crystalline structure, and the antibacterial effect is only effective under UV illumination owing to their large band-gap energy. In this paper, we have demonstrated a significantly enhanced antibacterial activity of hierarchical anatase TiO2 NFs against Staphylococcus aureus in the presence of UV light. Furthermore, the uniform deposition of a large quantity of Ag nanoparticles on the surface of the TiO2 NFs ensured a significant enhancement of the antibacterial performance, even under dark conditions. These results were obtained by exploiting the enhanced photocatalytic effect achieved through control of the crystallinity, as well as the enhanced surface area of the nanomaterials.