This study aimed to elucidate the antimicrobial efficacy of carboxymethylated β-glucan (CMG) against Staphylococcus aureus and the possible underlying mechanisms. β-Glucan (BG) was extracted from naked barley bran, then ultrafiltrated to obtain four fractions (BG-1, BG-2, BG-3, BG-4). Five types of CMG were prepared using an amidation reaction and characterized by FTIR spectroscopy. The underlying mechanisms were studied using changes in the transmembrane electrical potential, pH gradient, and intracellular ATP, confocal laser scanning microscopy, and electron microscopy. The data showed that CMG-2 had the highest antibacterial activity. CMG-2 induced membrane permeabilization, morphological changes, and structural disruption in S. aureus cells, with the formation of blebs and microspheres, the disruption of cell walls and membranes, and cell lysis. Our findings provide insight into the antimicrobial mechanism of CMG against S. aureus, which will provide a foundation for developing CMG as a novel antibacterial agent in the food and biomedical fields.