This review deals with a particular application of microfluidic devices such as fluids separation and water treatment. Through the different sections of the document, the reader can find not only a state of the art of specific applications related to fluids separation but also how this kind of micro-technology is being efficiently used by designing novel devices to carry out specific tasks of separation and water purification. In particular, this work has focused in three important processes: liquid–liquid separation; separation of biological samples; and water purification. After reading the document, the reader will have a clear view of how different microfluidic devices and technologies can be efficiently used in separation processes.