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Cueva del Viento, located in the Canary Islands, Spain, is the Earth's sixth‐longest lava tube, spanning 18,500 m, and was formed approximately 27,000 years ago. This complex volcanic cave system is characterized by a unique geomorphology, featuring an intricate network of galleries. Despite its geological significance, the geomicrobiology of Cueva del Viento remains largely unexplored. This study employed a combination of culture‐dependent techniques and metabarcoding data analysis to gain a comprehensive understanding of the cave's microbial diversity. The 16S rRNA gene metabarcoding approach revealed that the coloured microbial mats (yellow, red and white) coating the cave walls are dominated by the phyla Actinomycetota, Pseudomonadota and Acidobacteriota. Of particular interest is the high relative abundance of the genus Crossiella, which is involved in urease‐mediated biomineralization processes, along with the presence of genera associated with nitrogen cycling, such as Nitrospira. Culture‐dependent techniques provided insights into the morphological characteristics of the isolated species and their potential metabolic activities, particularly for the strains Streptomyces spp., Paenarthrobacter sp. and Pseudomonas spp. Our findings underscore the potential of Cueva del Viento as an ideal environment for studying microbial diversity and for the isolation and characterization of novel bacterial species of biotechnological interest. [ABSTRACT FROM AUTHOR]