Gram-negative bacteria release outer membrane vesicles (OMVs) with great potential in a variety of biomedical applications. The study aimed to investigate nutritional growth supplements (HBS, FBS, β-Cyclodextrin, or Cholesterol) and their impact on H. pylori’s OMVs biological properties. SDS-page showed a difference in protein components between all OMVs populations. Nuclear morphology for cells treated with 40 μg/mL of ExHp-CD displayed no change in nuclear morphology, while cells treated with ExHp-HBS, ExHp-FBS, and ExHp-Chlos showed chromatin condensation and nuclear fragmentation. CCK-8, LDH, and WST-1 assays showed no adverse effect for ExHp-CD in all used concentrations, while treated cells were negatively affected under higher concentrations for ExHp-HBS and ExHp-FBS (40 and 50 μg/mL) or in almost all the used concentrations for ExHp-Chlos. Obtained OMVs populations were phagocyted by macrophage RAW 264.7 cells leading to activate macrophages and produce cytokines. IL-10 and IL-4 were significantly (p< 0.05) increased after stimulation with all obtained OMVs, while levels of IFN-γ and IL-12 were not significantly increased. All OMVs populations increased CD206 ratio over CD86 ratio, demonstrating M2-phenotype polarization. Current findings revealed that growth supplements influenced OMVs biological properties while inducing Th2-immune response. This knowledge could determine which type of OMVs population for the required biomedical application.