Extracellular vesicles (EVs) are a heterogeneous group of membrane-encapsulated vesicles released by cells into the extracellular space. They play a crucial role in intercellular communication by transporting bioactive molecules such as proteins, lipids, and nucleic acids. EVs can be detected in body fluids, including blood plasma, urine, saliva, amniotic fluid, breast milk, and pleural ascites. The complexity and diversity of EVs require a robust and standardized approach. By adhering to standardized protocols and guidelines, researchers can ensure the consistency, purity, and reproducibility of isolated EVs, facilitating their use in diagnostics, therapies, and research. Exosomes and microvesicles represent an exciting frontier in modern medicine, with significant potential to transform the diagnosis and treatment of various diseases with an important role in personalized medicine and precision therapy. The primary objective of this review is to provide an updated analysis of the significance of EVs by highlighting their mechanisms of action and exploring their applications in the diagnosis and treatment of various diseases. Additionally, the review addresses the existing limitations and future potential of EVs, offering practical recommendations to resolve current challenges and enhance their viability for clinical use. This comprehensive approach aims to bridge the gap between EV research and its practical application in healthcare.