During recent years it has become increasingly clear that the release of extracellular vesicles (EVs) is a feature inherent to all cellular life forms. These lipid bilayer-enclosed particles are secreted by members of all domains of life: Eukarya, Bacteria and Archaea, being similar in size, general composition, and potency as a functional entity. Noticeably, the recent discovery of EVs derived from bacteria belonging to the Gram-positive phyla Actinobacteria and Firmicutes has added a new layer of complexity to our understanding of bacterial physiology, host interactions, and pathogenesis. Being nano-sized structures, Gram-positive EVs carry a large diversity of cargo compounds, including nucleic acids, viral particles, enzymes, and effector proteins. The diversity in cargo molecules may point to roles of EVs in bacterial competition, survival, material exchange, host immune evasion and modulation, as well as infection and invasion. Consequently, the impact of Gram-positive EVs on health and disease are being revealed gradually. These findings have opened up new leads for the development of medical advances, including strategies for vaccination and anti-bacterial treatment. The rapidly advancing research into Gram-positive EVs is currently in a crucial phase, therefore this review aims to give an overview of the groundwork that has been laid at present and to discuss implications and future challenges of this new research field.