Extremophiles are microorganisms that populate habitats considered inhospitable from an anthropocentric point of view and are able to tolerate harsh conditions such as high temperatures, extreme pHs, high concentrations of salts, toxic organic substances, and/or heavy metals. These microorganisms have been broadly studied in the last 30 years and represent precious sources of biomolecules and bioprocesses for many biotechnological applications; in this context, scientific efforts have been focused on the employment of extremophilic microbes and their metabolic pathways to develop biomonitoring and bioremediation strategies to face environmental pollution, as well as to improve biorefineries for the conversion of biomasses into various chemical compounds. This review gives an overview on the peculiar metabolic features of certain extremophilic microorganisms, with a main focus on thermophiles, which make them attractive for biotechnological applications in the field of environmental remediation; moreover, it sheds light on updated genetic systems (also those based on the CRISPR-Cas tool), which expand the potentialities of these microorganisms to be genetically manipulated for various biotechnological purposes.