Staphylococcus aureus is a pathogenic bacterium but also a commensal of skin and anterior nares in humans. As S. aureus transits from skins/nares to inside the human body, it experiences changes in temperature. The production and content of S. aureus extracellular vesicles (EVs) have been increasingly studied over the past few years, and EVs are increasingly being recognized as important to the infectious process. Nonetheless, the impact of temperature variation on S. aureus EVs has not been studied in detail, as most reports that investigate EV cargoes and host cell interactions are performed using vesicles produced at 37°C. Here, we report that EVs in S. aureus differ in size and protein/RNA cargo depending on the growth temperature used. We demonstrate that the temperature-dependent regulation of vesicle production in S. aureus is mediated by the alpha phenol-soluble modulin peptides (αPSMs). Through proteomic analysis, we observed increased packaging of virulence factors at 40°C, whereas the EV proteome has greater diversity at 34°C. Similar to the protein content, we perform transcriptomic analysis and demonstrate that the RNA cargo also is impacted by temperature. Finally, we demonstrate greater αPSM- and alpha-toxin-mediated erythrocyte lysis with 40°C EVs, but 34°C EVs are more cytotoxic toward THP-1 cells. Together, our study demonstrates that small temperature variations have great impact on EV biogenesis and shape the interaction with host cells. IMPORTANCE Extracellular vesicles (EVs) are lipid bilayer spheres that contain proteins, nucleic acids, and lipids secreted by bacteria. They are involved in Staphylococcus aureus infections, as they package virulence factors and deliver their contents inside host cells. The impact of temperature variations experienced by S. aureus during the infectious process on EVs is unknown. Here, we demonstrate the importance of temperature in vesicle production and packaging. High temperatures promote packaging of virulence factors and increase the protein and lipid concentration but reduce the overall RNA abundance and protein diversity in EVs. The importance of temperature changes is highlighted by the fact that EVs produced at low temperature are more toxic toward macrophages, whereas EVs produced at high temperature display more hemolysis toward erythrocytes. Our research brings new insights into temperature-dependent vesiculation and interaction with the host during S. aureus transition from colonization to virulence.