XTREMOLIFE will accelerate the bioprospection & biodiscovery of novel extremophile microorganisms. XTREMOLIFE uniqueness is advancing next-generation sampling technologies tailored for extreme conditions: [1] Enhanced Ferrybox for boats (self-operating), [2] novel XTREMOsensor for hand-held use, and [3] automated microscopic imagery identification. In addition, we explore (a large) biodiversity from 3 extremophilic ecosystems by bioprospecting across 5 regions (Route 1), and 4 untapped culture collections (Route 2). We will join those two routes by identifying promising extremophilic microorganisms, produced metabolites, elucidating their bioactivity and structure, optimizing their cultivation, and guaranteeing a baseline pathway towards full exploitation. We will focus on microalgae, cyanobacteria, and their associated microbiome, with a particular interest in bacteria and fungi. The culture collections NORCCA (Norway), ACUF (Italy), BEA (Spain), LEGE-CC (Portugal), will be prospected. In parallel, we will assess the microbiome and plan sampling from 5 locations: [1] Antarctic, [2] Volcanic aquatic environments in Canary Island, [3]Azores, [4]Mexico, and [5] Deserto of Taberna (Almería, Spain): focusing on hypersaline and extremely hot springs. We will assess the complexity of extremophilic ecology resorting to physical, chemical and biological data gathered. The chemical prospection will cover bioassays in the human, fish and plant health fields. Bioactive compounds will be isolated, and their structure fully characterized. Together, the consortium will select up to 7 different microbial compounds of interest-producing strains to be cultivated at lab scale, followed by a scale up to TRL 5 (feeding an Exploitation Roadmap). Finally, XTREMOLIFE will cover the legal framework of sampling, preserving and exploiting genetic material. The pathway towards full industrial exploitation (5-10 years after the project) in the various markets of applications will be characterized.