The studies of cave bacterial communities worldwide have revealed their potential to produce antibiotic molecules. In Costa Rica, ~400 caves have been identified; however, their microbial diversity and biotechnological potential remain unexplored. In this work, we studied the chemical composition and microbial diversity of a Costa Rican cave (known as the Amblipigida cave) located in Puntarenas, Costa Rica. Additionally, through culture-dependent methods, we evaluated the potential of its microbiota to produce antibiotic molecules. Mineralogical and elemental analyses revealed that the Amblipigida cave is primarily composed of calcite. However, small variations in chemical composition were observed as a result of specific conditions, such as light flashes or the input of organic matter. The 16S rRNA gene metabarcoding revealed an extraordinarily high microbial diversity (with an average Shannon index of ~6.5), primarily comprising bacteria from the phyla Pseudomonadota, Actinomycetota, Firmicutes and Acidobacteriota, with the family Pseudomonadaceae being the most abundant. A total of 93 bacteria were isolated, of which 15% exhibited antibiotic activity against at least one Gram-positive or yeast strain and were classified within the genera Lysobacter, Streptomyces, Pseudomonas, Brevundimonas and Bacillus. These findings underscore the highly diverse nature of cave microbiota and their significant biotechnological potential, particularly in the production of antibiotic compounds.