Secretion is an essential task for prokaryotic organisms to interact with their surrounding environment. In particular, the production of extracellular proteins and peptides is important for many aspects of an organism’s survival and adaptation to its ecological niche. In recent years, considerable progress has been made towards elucidating the structural and molecular mechanisms of the six secretion systems (types I–VI) of Gram-negative bacteria, the unique mycobacterial type VII secretion system, the General Secretory Pathways (Sec) and twin-arginine translocation (Tat). These advances have greatly enhanced our understanding of the complex mechanisms that these macromolecular structures use to deliver proteins and DNA into the extracellular environment or into target cells. In addition The rise of multidrug resistant bacteria is a major worldwide health concern. There is currently an unmet need for the development of new and selective antibacterial drugs. In this Review, we explore the structural and mechanistic relationships between these general, single- and double-membrane-embedded systems, and we briefly discuss how this knowledge can be exploited for the development of new antimicrobial strategies.