Bacterial and viral infections pose persistent threats to global public health. As core mediators of host-pathogen interactions, extracellular vesicles (EVs) exert bidirectional regulation over infectious progression by mediating immune defense and pathogenic injury. This review systematically elaborates the biological characteristics of host-derived, bacterium-secreted and virus-hijacked EVs, discusses their offensive-defensive crosstalk during infection, and summarizes their translational potential in non-invasive diagnosis, targeted therapy and vaccine development. Meanwhile, major obstacles restricting clinical translation, including the absence of standardized preparation protocols, suboptimal targeting performance and insufficient data on long-term biosafety, are critically assessed. Deciphering the bidirectional regulatory mechanisms of EVs holds the potential to overcome the limitations of traditional anti-infection regimens and provide novel insights for the precision medicine in infectious diseases.