The Sahara Desert, located at the northern part of the African continent, is one of the most inhabitable and harsh ecosystems on earth. In Tunisia, more than 40% of the country area is covered by the Sahara Desert, as well as a variety of extreme environments, which include many coastal natural saline systems, named locally as “Sebkha” and “Chotts.” Microbial communities, able to withstand extreme saline environments, have the capacity to regulate the osmotic pressure to resist the denaturing effects of salt. In addition, a common phenomenon in these environments is the occurrence of gradients in salinity as a result of evaporation, and halophilic microorganisms that are able to rapidly adjust their osmotic equilibrium as required when the outside salinity is changed. These gradients also result in the establishment of very diverse microbial compositions and structures, in which halophilic and halotolerant microorganisms from three domains of life (Eukarya, Bacteria, and Archaea) can thrive. Eukarya are a minority in high-salt environments, mostly represented by halotolerant Ascomycetes genera, such as Cladosporium, Alternaria, Aspergillus, Penicillium, Ulocladium, and Engyodontium. Salt-adapted bacteria are spread over a large number of phylogenetic groups (Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria, as well as Firmicutes and Actinobacteria), and the majority are moderate rather than extreme halophiles. The extreme hypersaline systems are dominated by the Archaea, and they often display a bright red color as a result of the large number of pigmented haloarchaea. These extremophilic microbes can lead to biotechnological applications for new metabolite production, plant protection, insect biocontrol, and fertility improvements in agriculture. Thus, they can help in promoting the development of a modern and competitive bioeconomy in arid regions. This chapter describes the microbial diversity in desert environments and saline systems of southern Tunisia using culture-independent and -dependent approaches. Relevant examples of extremophilic microorganisms from the desert with high biotechnological and industrial potential are also provided.