Berberine is a plant alkaloid with antibacterial, anti-inflammatory, anti-cancer, and hypolipidemic activities, etc. However, the application of berberine in pharmaceuticals has still limited due to its less solubility in water and poor bioavailability. In this study, to improve the bioavailability and increase the potential application of berberine in medicine, berberine nanoparticles were fabricated by roll milling method with the support of zirconium balls. The characterisation of berberine nanoparticles was evaluated by Field-Emission Scanning Electron Microscopy (FE-SEM), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and X-ray diffraction (XRD). The anti-Candida albicans activity was tested in-vitro. The results showed that the average particle size of berberine nanoparticles was about 60 nm after 120 hours roll milling and the minimal inhibitory concentration on Candida albicans was 1024 µg/ml.