Abstract: A new carbon dots with antibacterial properties was synthesized using Robusta coffee beans as raw materials, and its antibacterial mechanism against Staphylococcus aureus was explored. Robusta coffee carbon dots was synthesized via a one-step hydrothermal method and characterized using transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), Fourier transform infrared absorption spectroscopy (FTIR), Zeta potential, UV spectroscopy, and fluorescence spectroscopy. The antibacterial activity of Robusta coffee carbon dots against Staphylococcus aureus was evaluated by minimum inhibition concentration (MIC) and time-kill curve, while the antibacterial mechanism was investigated using scanning electron microscopy (SEM), TEM, and fluorescence microscopy. The results indicated that Robusta coffee carbon dots exhibited good dispersibility with negative charges and abundant surface functional groups such as O-H, C-H, and C-O. Furthermore, the MIC value of Robusta coffee carbon dots against Staphylococcus aureus was 250 μg/mL, effectively inhibiting bacterial growth within 8 hours by damaging the cell membrane of Staphylococcus aureus leading to leakage of intracellular substances and promoting the production of reactive oxygen species (ROS). In summary, the Robusta coffee carbon dots exhibited considerable antibacterial properties against Staphylococcus aureus, which was potential to be applicated in the prevention and control of Staphylococcus aureus to reduce the safety issues.