Clarifying the antibacterial mechanism of silver (Ag)-based materials is of great significance for the rational design, synthesis, and evaluation of antimicrobials. Herein, detailed description of the antibacterial mechanism of a synthesized silver deposited fullerene material (Ag(I)–C60) towards Staphylococcus aureus was surveyed from the point of view of DNA damage by ultraviolet–visible spectroscopy (UV–vis), inductively coupled plasma mass spectrometry (ICP-MS), and liquid chromatography-mass spectrometry (LC-MS). The model material, Ag(I)–C60, was prepared by liquid-liquid interfacial precipitation method, and characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), thermos-gravimetric analysis (TGA), and nitrogen adsorption/desorption analysis. Ultra-efficient bacteriostatic rate of Ag(I)–C60 was found to be 88.98% under light irradiation for 20 min. UV–vis measurement of the composition changes of four DNA bases showed that they changed in the presence of Ag(I)–C60 under light irradiation, suggesting Ag(I)–C60 could destroy the cells and genetic material of Staphylococcus aureus and thereby inhibit its growth and reproduction. ICP-MS analysis demonstrated the releasing behavior of Ag+ from Ag-based materials. Finally, the transformation pathway of G, A, C, and T were measured by LC-MS, demonstrating the conversion of Adenine (m/z 136.06) to 8-OH-Ade (m/z 174.04). These collective results suggested that Ag(I)–C60 was a new ultra-efficient antibacterial by slowly releasing Ag+ in water and producing a large amount of ROS under light.