Extracellular vesicles (EVs) derived from Staphylococcus aureus (S. aureus) carry multiple components, such as toxins, antigens, and resistance determinants, whose production is influenced by exposure to β-lactam antibiotics. However, systematic studies on the effects of β-lactam antibiotics on the release and functions of EVs remain limited. In this study, we isolated EVs from an OS-MRSA (OS-200) strain by ultracentrifugation, including both EVs untreated with antibiotics and those exposed to sub-inhibitory concentrations of oxacillin (OI-EVs). Exposure to sub-inhibitory concentrations of oxacillin (half and one-eighth of the minimum inhibitory concentration [MIC]) significantly enhanced the secretion of OI-EVs and increased the abundance of several proteins, including penicillin-binding protein 2, EmrB, Hlg, and enolase, in a concentration-dependent manner. Notably, EV1/2MIC exhibited more pronounced functional changes and enhanced hemolytic activity against rabbit red blood cells and promoted biofilm formation by OS-200. Additionally, OI-EVs stimulated the secretion of interleukin-6 and tumor necrosis factor-α by THP-1 macrophages in a dose-dependent manner and demonstrated greater penetration potential into A549 lung epithelial cells. Exposure to sub-inhibitory concentrations of oxacillin significantly altered the secretion and composition of EVs, highlighting a novel relationship between antimicrobial exposure, exosome biogenesis, and OS-MRSA pathogenicity. These findings provide new insights into how β-lactam antibiotics influence host–pathogen interactions by modulating the secretion of bacterial vesicles.