Bacterial and human cells produce extracellular vesicles (EVs) in response to diverse stimuli, e.g., toxins, oxidative stress, nutrient depletion, or high cell density. Here, we describe a cocultivation platform that allows recovery of mixed extracellular vesicles (mix-EVs) produced simultaneously by both cell types. We investigated interactions between Grampositive and Gram-negative bacteria (Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli, and Neisseria meningitidis) and human peripheral blood mononuclear cells (PBMCs). The production of the mix-EVs population decreased with higher bacterial concentrations. Exposing PBMCs to mix-EVs repressed the general transcriptomic signature, in contrast with a significant upregulation generated by single bacterial-EVs. However, mix-EVs-derived IL-1β upregulation was confirmed at the protein level. Inhibition experiments showed that IL-1β production involved TLR2 and TLR4 signaling, acting through IRAK-1 and TRAF6 related pathways. This approach provides a new platform for the study of EVs at the pathogen−host interface and presents mechanistic insights into the effect of EVs on an infected host.