The objective of the present study was to reveal the antibacterial mechanism of lactobionic acid (LBA) against methicillin-resistant (MRSA) using quantitative proteomics by sequential window acquisition of all theoretical mass spectra (SWATH-MS) to analyze 100 differentially expressed proteins after LBA treatment. Furthermore, multiple experiments were conducted to validate the results of the proteomic analysis including reactive oxygen species (ROS), virulence-associated gene expression, and the relative quantification of target proteins and genes by parallel reaction monitoring and quantitative real-time PCR. Combining the ultrastructure observations, proteomic analysis, and our previous research, the mode of LBA action against MRSA was speculated as cell wall damage and loss of membrane integrity; inhibition of DNA repair and protein synthesis; inhibition of virulence factors and biofilm production; induction of oxidative stress; and inhibition of metabolic pathways. These results suggest potential applications for LBA in food safety and pharmaceuticals, considering its multitarget effects against MRSA.