Abstract: As a special habitat with long-term dark, moist and nutritive deficiency, the cave contains rich microbial groups with unique metabolic types. In this study, 623 bacterial strains were isolated from three substrates soil, stalactite and water collected from Tiandong Cave in Guizhou Province, and these bacteria were included into 228 species of 89 genera. Of them, actinobacteria, alphaproteobacteria and gammaproteobacteria were the dominant groups which had the strain proportion of 50.72%, 18.45% and 16.85%, respectively. On genus level, Pseudomonas (14.45%), Microbacterium (12.20%) and Streptomyctes (9.31%) were the dominant. Substrates of soil, stalactite and water had their dominant bacterial genera respectively as Streptomyces (13.72%), Microbacterium (15.44%) and Caulobacter (18.95%).The Shannon and Margalef indexes of bacteria in soil were the highest, while that in water were the lowest. Of 623 bacteria isolates, 86 species could produce amylase, 64 species could produce cellulase, and 79 species could produce proteinase. Strains from soil with activities of amylase and cellulase were more than that from stalactite and water, while the strains from water with cellulase activity were more than that from the rest substrates.