Bacterial biofilms are associated clinically with many bacterial infections including those caused by bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus. In recent years, extracellular vesicles produced by bacteria have been isolated from biofilm communities. Vesicles have been described in depth and can encapsulate various virulence factors including toxins and immunomodulatory compounds. Vesicles may be important for virulence and survival by serving as a vehicle for the secretion and concentrated delivery of these molecules. Studying extracellular vesicles is an important step towards understanding biofilm formation, structure, and disruption with the ultimate goal of preventing or treating hospital infections caused by bacterial pathogens residing in biofilms. Here we describe the protocol for isolating vesicles from biofilm produced by Bacillus subtilis.