Bacterial vesicles (BVs) are membrane-bound extracellular vesicles (EV) released from bacteria. They are known to play crucial role in bacterial communication, host-pathogen interactions, transfer of virulence factors, contribute to immune modulation and are the key players in microbial pathogenesis and survival in the host. Despite their significance, isolation and investigating BVs from human samples remains challenging, necessitating an easy, reliable and reproducible protocol. BVs have been limited due to methodological difficulties in isolating them from host-derived EVs, and the existing knowledge primarily relies on bacteria cultured under controlled laboratory conditions. This study presents a method, where we can identify the enriched BVs and characterizing them from plasma and stool samples of healthy individuals. Blood and fecal samples were collected, processed to density gradient ultracentrifugation to isolate and enrich BVs. Morphological characterization was performed using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA). Further, molecular markers OmpA (BV marker) was used to differentiate from host EVs (Alix as marker) using Western blot. Further the BV fraction was analyzed for LPS and LTA using ELISA. To understand functional relevance, BVs proteomics was performed from BV enriched plasma and stool using mass spectrometry from healthy individuals. The enriched BVs were also co-cultured with healthy peripheral blood mononuclear cells, labelled with Pkh26 dye and analysed at different time points for mRNA expression of candidate genes involved in immune regulation by qRT-PCR. Both TEM and NTA confirmed the presence of BVs, with sizes ranging from 25 nm to 250 nm. The western blot analysis revealed the fractions 6-9 are enriched with host EVs with the presence of Alix and fractions 10-13 contains BVs with the presence of OmpA. Interestingly, the proteomic analysis identified 439 proteins associated with plasma-derived BVs and 327 in stool-derived BVs, with 300 common to both. The Gene ontology and KEGG pathway analysis revealed the majority of proteins associated were immune regulation, cell activation, binding, and catalytic activity. Next, the functional assays indicated BVs were uptaken by PBMCs within 10 mins and it upregulated Toll-like receptor 2 (TLR-2) expression within 30 min. Hence, study establishes a reliable method to identify enriched BV population from human samples. Revealed the proteins associated with BVs in healthy individuals and their role in immune regulation. These findings may provide a platform to investigate BVs potential for diagnostic and therapeutic applications in various diseases.