Extreme aquatic environments account with a vast and yet largely unexplored biodiversity that holds immense potential for novel active biomolecules with multiple industrial applications. However, the harsh and challenging conditions such as accessibility, environmental hazards, ineffective or inaccurate sampling techniques, and poor bioprospecting pose significant obstacles to their exploration and sampling. Overcoming these challenges is crucial for unlocking the potential of extremophiles to benefit society. The EXPLORA project will depart from two extreme aquatic ecosystems selected such as the acidic Rio Tinto in Spain and the psychrophilic Antarctic Region. By developing innovative, accurate, and sustainable tailor-made sampling methods, employing cutting-edge bioprospecting techniques, and utilising state-of-the-art analytical tools for isolation of bioactive substances, in the EXPLORA project it is expected to find and isolate microorganisms which produce novel antimicrobial and antioxidants compounds, sialylated extracellular polymeric substances and, plastic-degrading acidophilic enzymes which applicability will be validated for pharmaceutical, cosmetic, nutraceutical, and for PET recycling purposes, respectively. Additionally, deeper understanding on the legal frameworks in the planned contexts such as the Nagoya Protocol and Antarctic Treaty Legal Requirements is also envisaged. By achieving these objectives, EXPLORA will contribute to the sustainable exploration of biodiversity hotspot regions, advancing the development of next-generation sampling methods, expanding bioprospecting from the screening for new chemicals into biological function, and contributing to the understanding of potential trade-offs inherent in the exploitation of ocean and other aquatic biodiversity.