This work focuses on rock-inhabiting fungi (RIF) of Antarctic rocky deserts, considered the closest to a possible Martian habitat, as the best example of adaptation to the extremes. The study of RIF ecophysiology, resistance and adaptation provides tools that shed light on the evolution of extremophily. These studies also help define the actual limits for life and provide insight for investigating its existence beyond our planet. The scientific results obtained from over 20 years of research on the biodiversity, phylogeny and evolution toward extremotolerance reviewed here demonstrate how these fascinating organisms can withstand conditions well beyond those in their natural environment. A final focus is given on results and perspectives arising from a recent proteomic approach, and from astrobiological experiments and their significance for future space exploration. These studies demonstrate that Antarctic RIF offer an excellent opportunity to investigate many basic, but also applicative areas of research on extremophily.