We report the discovery and detailed analysis of an extraordinary colonial spider assemblage in Sulfur Cave, a chemoautotrophic sulfidic ecosystem located on the Albania-Greece border. The colony, comprising an estimated 69,000 individuals of Tegenaria domestica (Agelenidae) and more than 42,000 of Prinerigone vagans (Linyphiidae), spans a surface area of over 100 m²—representing the first documented case of colonial web formation in these species. Stable isotope analyses (δ¹³C and δ¹⁵N) revealed that the trophic web sustaining this assemblage is fueled by in situ primary production from sulfur-oxidizing microbial biofilms then transferred through chironomid larvae and adults to higher trophic levels. Morphological and molecular data confirmed the identity of the two spider species and revealed that their populations in Sulfur Cave are genetically distinct from other populations. Regarding T. domestica, we found a seasonal pattern in fecundity, with significantly larger egg clutches in early summer. Microbiome analysis of this species also revealed a lower Shannon diversity in the cave population compared with a surface individual captured nearby. Our findings unveil a unique case of facultative coloniality in this cosmopolitan spider, likely driven by resource abundance in a chemoautotrophic cave, and provide new insights into the adaptation and trophic integration of surface species in sulfidic subterranean habitats.