The deep, hot subsurface biosphere represents one of the largest biomes of our planet. Recent scientific explorations have confirmed that in spite of a multitude of physical and chemical extremities, the deep biosphere could harbor up to 19% of Earth’s total biomass. Evolution, adaptation, and metabolic abilities of these extremophilic microorganisms intrigued us to answer the most fundamental questions about origin, evolution, limits, and mechanisms of adaptation of life on this planet and their exploration in other planetary bodies. This chapter presents a comprehensive review of: (a) general attributes of extremophiles; (b) deep subsurface as the hotspot for extremophiles and astrobiological research; and (c) astrobiological targets with subsurface analogues on Earth and role of bioenergetics model for predicting habitability. Overall, this review seeks to synthesize the existing state of knowledge on the topic and provides a direction for future research and applications in astrobiology.