The application of modern advanced techniques in molecular biology is revealing unexpectedly high levels of microbial diversity and complexity. However, the invisible loss of microbial diversity in the environment deriving, for example, from global changes and anthropogenic activities, is not really perceived. In this context, culture collections worldwide have become a valuable resource for the sustainable use of microbial diversity and its conservation. They provide pure cultures and genetic materials that are required for a number of research and teaching purposes, as well as for bioprospecting aims and their subsequent exploitation in biotechnological fields. This Special Issue has been launched with the aim of showcasing the diversity and biotechnological potential of microorganisms (e.g., Bacteria, Archaea, cyanobacteria, microalgae, fungi, yeasts, and protozoa) belonging to culture collections kept worldwide.