Gram-negative and Gram-positive bacteria release a variety of membrane vesicles through different formation routes. Knowledge of the structure, molecular cargo and function of bacterial extracellular vesicles (BEVs) is primarily obtained from bacteria cultured in laboratory conditions. BEVs in human body fluids have been less thoroughly investigated most probably due to the methodological challenges in separating BEVs from their matrix and host-derived eukaryotic extracellular vesicles (EEVs) such as exosomes and microvesicles. Here, we present a step-by-step procedure to separate and characterize BEVs from human body fluids. BEVs are separated through the orthogonal implementation of ultrafiltration, size-exclusion chromatography (SEC) and density-gradient centrifugation. Size separates BEVs from bacteria, flagella and cell debris in stool; and blood cells, high density lipoproteins (HDLs) and soluble proteins in blood. Density separates BEVs from fibers, protein aggregates and EEVs in stool; and low-density lipoproteins (LDLs), very-low-density lipoproteins (VLDLs), chylomicrons, protein aggregates and EEVs in blood. The procedure is label free, maintains the integrity of BEVs and ensures reproducibility through the use of automated liquid handlers. Post-separation BEVs are characterized using orthogonal biochemical endotoxin and Toll-like receptor-based reporter assays in combination with proteomics, electron microscopy and nanoparticle tracking analysis (NTA) to evaluate BEV quality, abundance, structure and molecular cargo. Separation and characterization of BEVs from body fluids can be done within 72 h, is compatible with EEV analysis and can be readily adopted by researchers experienced in basic molecular biology and extracellular vesicle analysis. We anticipate that this protocol will expand our knowledge on the biological heterogeneity, molecular cargo and function of BEVs in human body fluids and steer the development of laboratory research tools and clinical diagnostic kits.