Bioactive molecules and immune factors in the bovine colostrum (BC) are important elements of passive immunity that prevent bacterial infection. However, the mechanisms underlying the antimicrobial activity of BC are not fully understood. We assessed the antibacterial properties of BC-derived exosomes (BC-Exo) and found that they had bacteriostatic, anti-hemolytic, and biofilm-eradication effects on Staphylococcus aureus. Moreover, cell surface deformation and reduced ATP production were observed following BC-Exo treatment. The most reasonable explanation for this finding is that BC-Exo has a strong inhibitory effect on the oxidative phosphorylation pathway in S. aureus. We demonstrated, for the first time, that BC-Exo can exhibit clear antimicrobial activity against S. aureus. Our findings constitute an important basis for future antibiotic discovery.