In this study, novel antibacterial hemp fibres grafted with quaternary ammonium groups, named HF-GTA, were prepared by alkalisation, oxidation, amination, and quaternisation multistage reactions. The chemical structure and micromorphology of the fibres were characterised by Fourier-transform infrared spectroscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, thermogravimetric analysis, and X-ray diffraction. The grafting and reaction mechanisms were proved to be successful, which indicated that the grafting reaction primarily occurred on the hydroxyl groups of cellulose and hemicellulose in the hemp fibres, where they retained good fibrous morphology, thermal stability and hygroscopicity. The antibacterial activity of HF-GTA exhibited the best antibacterial activity, where the antibacterial ratios against E. coli and S. aureus were 95.41% and 99.64%, respectively. Even after 30 times of washing, the antibacterial activity was retained at 89.78% and 91.12%, indicating that HF-GTA was endowed with good washing resistance. The antibacterial mechanism occurred because the electrostatic reaction reduced the electrochemical potential on the cell membrane, leading to the release of cytoplasmic substances and the dissolution of cells, thereby producing antibacterial activity. It would be significantly important to guarantee textile quality and prevent disease transmission.