The article reviews the production and functions of extracellular vesicles (EVs) in Gram-positive bacteria, mycobacteria, and fungi. EVs are produced by all domains of life and play crucial roles in microbial physiology and pathogenesis. In Gram-negative bacteria, EVs are formed by the pinching off of the outer membrane, but the mechanism for their release in thick-walled organisms like Gram-positive bacteria, mycobacteria, and fungi remains unknown. Despite this, EVs have been detected in these organisms, containing various cargo such as nucleic acids, toxins, lipoproteins, and enzymes. The review discusses the current understanding of vesiculogenesis in thick-walled microorganisms and highlights the diverse cargo and functions associated with EVs in these species. It also explores the potential mechanisms by which EVs can traverse the thick cell walls, including turgor pressure, cell wall-modifying enzymes, and protein channels or structural cables. The article further examines the role of EVs in virulence, immune evasion, and interspecies communication, emphasizing their significance in microbial pathogenesis.The article reviews the production and functions of extracellular vesicles (EVs) in Gram-positive bacteria, mycobacteria, and fungi. EVs are produced by all domains of life and play crucial roles in microbial physiology and pathogenesis. In Gram-negative bacteria, EVs are formed by the pinching off of the outer membrane, but the mechanism for their release in thick-walled organisms like Gram-positive bacteria, mycobacteria, and fungi remains unknown. Despite this, EVs have been detected in these organisms, containing various cargo such as nucleic acids, toxins, lipoproteins, and enzymes. The review discusses the current understanding of vesiculogenesis in thick-walled microorganisms and highlights the diverse cargo and functions associated with EVs in these species. It also explores the potential mechanisms by which EVs can traverse the thick cell walls, including turgor pressure, cell wall-modifying enzymes, and protein channels or structural cables. The article further examines the role of EVs in virulence, immune evasion, and interspecies communication, emphasizing their significance in microbial pathogenesis.