Natural berberine-hybridized benzimidazoles as potential antibacterial agents were constructed to treat Staphylococcus aureus infection in the livestock industry. Bioassay showed that some new berberine-benzimidazole hybrids exhibited potent antibacterial efficacies, especially, the 2,4-dichlorobenzyl derivative 7d not only showed strong activity against S. aureus ATCC 29213 with the MIC value of 0.006 mM but also effectively eradicated bacterial biofilm and exhibited low toxicity toward mammalian cells. The drug combination experiments showed that compound 7d together with norfloxacin could enhance the antibacterial efficacy. Moreover, the 2,4-dichlorobenzyl derivative 7d did not show obvious propensity to develop bacterial resistance. Preliminary mechanism studies revealed that the active molecule 7d could damage the membrane integrity, stimulate ROS generation, and bind with DNA as well as S. aureus sortase A, thus exerting powerful antibacterial ability. In light of these facts, berberine-benzimidazole hybrid 7d showed a large potentiality as a new bactericide for treating S. aureus in the livestock industry.