Known　as　the　largest　negative　terrain　on　the　earth＇s　surface,　the　karst　tiankeng　is　isolated　by　vertical　cliffs　and　maintains　a　unique　ecosystem　within　it.　However,　knowledge　of　the　microbial　ecology　of　karst　tiankengs　does　not　match　their　importance.　To　this　end,　we　conducted　extensive　sampling　across　16　karst　tiankengs　and　2　sites　and　used　high-throughput　sequencing　methods　to　analyze　soil　bacteria　and　fungi.　This　study　found　abundant　and　diversified　microbial　communities　in　karst　tiankengs,　with　Proteobacteria,　Acidobacteriota　(Bacteria),　and　Ascomycota,　Basidiomycota　(Fungi)　being　the　main　phyla.　Compared　to　the　degraded　karst　landscape,　karst　tiankengs　maintained　a　higher　richness　of　bacterial　taxa.　The　common　and　exclusively　microbial　taxa　in　karst　tiankengs　follow　copiotrophic　and　opportunistic　strategies,　and　these　taxa　are　associated　with　high　soil　nutrients　(TN　and　SOM)　and　water　content.　Karst　tiankeng　maintained　a　stable　microbial　network　structure　through　cooperation,　and　the　dispersal　limitation　dominated　the　bacterial　and　fungal　community　assembly.　Our　results　enhance　the　understanding　of　microbiomes　in　subterranean　karst　ecosystems,　highlighting　that　karst　tiankengs　are　important　refuges　for　microbial　diversity　in　degraded　karst　landscapes.　©　2025　John　Wiley　&　Sons　Ltd.