<p>This study investigated the prokaryotic microbiota-comprising both archaea and bacteria-of Manjanggul Cave, a UNESCO World Heritage lava tube on Jeju Island, South Korea. Despite their geological significance, the archaeal and bacterial community structures within Jeju Island’s diverse cave systems have remained largely unexplored, primarily due to access restrictions. Here, we used high-throughput sequencing of archaeal and bacterial 16S rRNA genes, as well as biostatistics, to elucidate the structure and diversity of these communities within Manjanggul Cave. The findings revealed that the bacterial community was dominated by Proteobacteria, while Crenarchaeota (classified by silva) was the predominant archaeal phylum. Notably, the ammonia-oxidizing archaeal genus <i>Nitrososphaera</i> was highly abundant, whereas ammonia-oxidizing bacteria (genera <i>Nitrosococcus</i> and <i>Nitrosomonas</i>) were detected at extremely low levels. The nitrite-oxidizing bacterial genus <i>Nitrospira</i> was consistently abundant across all samples, suggesting that <i>Nitrososphaera</i>/<i>Nitrospira</i> may play crucial roles in aerobic nitrification within the cave ecosystem. Intriguingly, a high proportion of haloarchaea was also observed, which may be attributed to the unique geological characteristics of Jeju Island, indicating a distinct microbial ecosystem within Manjanggul Cave. In conclusion, this study provides molecular evidence for the mixing of indigenous cave-associated prokaryotic communities with external microbial populations, suggesting potential alterations in the composition of both communities within Manjanggul Cave. These findings underscore the importance of research to understand the impact of human activities on cave microbial ecosystems, and the need for conservation strategies to preserve these unique environments.</p>