Deep hypersaline anoxic basins (DHABs) are marine extreme habitats, firstly discovered in the 1970s of the last century, located in several oceanographic regions, including the Mediterranean and Red Sea and the Gulf of Mexico. These basins are filled with brines that do not mix with the overlying seawater, due to a density difference. Brine and seawater result separated by a thick interface acting as a trap for particulate and cells. Some microbiological studies focused on seawater-brine interfaces of DHABs, showing that microbial populations are differentially distributed according to the gradient of salinity, oxygen, and nutrients occurring in such transition zones. Moreover, DHABs’ brines were intensively studied showing that specific bacterial, archaeal, and eukaryotic populations thrive there. In the last few years, cultivation and “omics”-based approaches have been used with samples collected from DHABs around the world, allowing clarifying metabolic processes of paramount ecological importance and pointing out the high biotechnological potential of the inhabiting extremophiles.