Eukaryotic cells communicate with one another through multiple pathways. An established route of communication between eukaryotic cells is via the production of a range of different membrane bound signalling “packages”, called extracellular vesicles (EVs). EVs are produced by all domains of life and carry proteins, nucleic acid (RNA and DNA), and other biological material, travelling between cells and around the body to deliver a range of chemical messages. Bacteria can also produce EVs that communicate with each other to co-ordinate population behaviour, as well as with eukaryotic cells to stimulate host defence or induce tolerance. Here I investigate the poorly explored axis where EVs are the vehicle for communication between eukaryotic cells and bacteria. As a first step, I have isolated EVs from tissue cultured eukaryotic cells grown in Advanced RPMI media with minimal EV-depleted FBS. NanoEVs were isolated from spent culture media using sequential centrifugation (2,000 × g, 10,000 × g) and concentration (100 kDa filter) before purifying using size exclusion chromatography columns. NanoEV-rich fractions were pooled based on particle (Nanoparticle Tracking Analysis) and protein quantity data. NanoEVs were characterised by electron microscopy and expression of exosomal markers. Eukaryotic nanoEVs were then characterised in their effect upon the growth of Escherichia coli as a model bacterium, also grown in tissue culture media to mimic relevant in vivo conditions.