Pathogenic bacteria can cause several millions of deaths per year, understanding their mechanisms of infection to allow the treatment and prevention of disease has become a worldwide research priority. It is now well established that Extracellular Vesicles (EVs) are secreted by virtually every living cell, including prokaryotes. Community accepted guidelines are provided for the study of human EVs, but not for the equivalent investigations in bacteria. The population of EVs released from each bacterium is highly heterogeneous in size, structure and molecular content. We have tested two commonly used EV isolation methods in different microorganisms, Density Gradient Centrifugation (DGC) and Size Exclusion Chromatography (SEC). Results indicate that each method has own advantages which are directly linked to the downstream application of interest with the isolated EVs. Pathogenic bacteria are thought to use EVs with diverse roles in the infection, thus we have also characterised the molecular cargo of EVs released by different pathogens and non-pathogenic bacteria, with the aim of finding signature molecules with a potential relevance in infection. Each microorganism secretes a specific EV composition profile which strongly depends on the culture conditions. Regarding bacteria-host interaction, we have confirmed bacterial EVs enter relevant host cells in vitro using microscopy, with the majority localising to the cytoplasm. Host cells also display a signature transcriptomic response to bacterial EVs. Overall, we suggest comprehensive reporting of bacterial EVs isolation procedures, culture conditions and meaningful controls to allow significant comparisons and conclusions in such a novel, heterogeneous and yet understudied field.