Escherichia coli A0 34/86 (EcO83) is a probiotic bacterium that is used in newborns to prevent nosocomial infections and diarrhoea. Mechanistically, EcO83 enhances innate immunity by inducing key inflammatory cytokines in vitro, and when administered intranasally, it modulates the recruitment of cells to the lung. This study investigates the immunomodulatory effects of EcO83-derived extracellular vesicles (EcO83-EVs) as a safer alternative to live bacteria. We isolated, purified, and characterised EcO83-EVs following the MISEV guidelines. The proteomics data of the EVs were compared with the proteomics data of cell extracts of the EcO83.