Extracellular vesicles (EVs) are produced by all domains of life. While Gram-negative bacteria produce EVs by pinching off their outer membrane, the mechanism by which Gram-positive bacteria, mycobacteria, and fungi release EVs through their thick cell walls remains unclear. EVs from these organisms contain various cargoes, including nucleic acids, toxins, lipoproteins, and enzymes, and play important roles in microbial physiology and pathogenesis. This review discusses current research on EV production in thick-walled microorganisms, focusing on their cargo and functions. EVs are important for microbial pathogenesis, including the secretion of virulence factors, immune modulation, and the transfer of pathogens. In Gram-negative bacteria, EVs are called outer-membrane vesicles (OMVs) and contain virulence factors, adhesins, DNA, RNA, and toxins. OMVs can contribute to microbial pathogenesis and have been used in therapeutic vaccines. In Gram-positive bacteria, mycobacteria, and fungi, EVs are produced through mechanisms that are not fully understood, but they are involved in cell-to-cell communication, nutrient acquisition, and immune evasion. EVs from Gram-positive bacteria, mycobacteria, and fungi have been isolated and characterized, and their cargo includes proteins, nucleic acids, and lipids. EVs can act as vehicles for the secretion of virulence factors and can deliver these factors directly to host cells. EVs also play a role in biofilm formation and in the pathogenesis of fungal infections. The release of EVs from the cell wall is a complex process, and several hypotheses have been proposed to explain how EVs escape the thick cell walls of these organisms. EVs are important for microbial survival and pathogenesis, and their study has led to new insights into microbial physiology and pathogenesis. The production and function of EVs in Gram-positive bacteria, mycobacteria, and fungi are areas of active research, with many questions remaining to be addressed. Understanding the mechanisms of EV production and function in these organisms is important for the development of new therapies and vaccines.Extracellular vesicles (EVs) are produced by all domains of life. While Gram-negative bacteria produce EVs by pinching off their outer membrane, the mechanism by which Gram-positive bacteria, mycobacteria, and fungi release EVs through their thick cell walls remains unclear. EVs from these organisms contain various cargoes, including nucleic acids, toxins, lipoproteins, and enzymes, and play important roles in microbial physiology and pathogenesis. This review discusses current research on EV production in thick-walled microorganisms, focusing on their cargo and functions. EVs are important for microbial pathogenesis, including the secretion of virulence factors, immune modulation, and the transfer of pathogens. In Gram-negative bacteria, EVs are called outer-membrane vesicles (OMVs) and contain virulence factors, adhesins, DNA, RNA, and toxins. OMVs can contribute to microbial pathogenesis and have been used in therapeutic vaccines. In Gram-positive bacteria, mycobacteria, and fungi, EVs are produced through mechanisms that are not fully understood, but they are involved in cell-to-cell communication, nutrient acquisition, and immune evasion. EVs from Gram-positive bacteria, mycobacteria, and fungi have been isolated and characterized, and their cargo includes proteins, nucleic acids, and lipids. EVs can act as vehicles for the secretion of virulence factors and can deliver these factors directly to host cells. EVs also play a role in biofilm formation and in the pathogenesis of fungal infections. The release of EVs from the cell wall is a complex process, and several hypotheses have been proposed to explain how EVs escape the thick cell walls of these organisms. EVs are important for microbial survival and pathogenesis, and their study has led to new insights into microbial physiology and pathogenesis. The production and function of EVs in Gram-positive bacteria, mycobacteria, and fungi are areas of active research, with many questions remaining to be addressed. Understanding the mechanisms of EV production and function in these organisms is important for the development of new therapies and vaccines.