The Xifeng Hot Spring is one of the eight largest hot springs in China, which is rich in radon gas and sulphur in karst scenery. Little is known about the microbiota structure in the spring. The water was collected from three sites containing the outlet of spring water discharge site (OWD), spring pool for tourist (SPT) and sewage effluent pool (SEP) in the Xifeng Hot Spring and further analyzed by culture-independent technique and culture-dependent method. A total of 57 phyla were identified from the water samples. The dominate phyla at OWD was Bacteroidetes (46.93%), while it was Proteobacteria in both sites of SEP and SPT with relative richness of 61.9% and 94.9%, respectively. Two bacteria, Deinococcus and Hymenobacter, that confirmed to be radiation-resistant, seven sulphur bacteria and three thermophilic bacteria were detected from Xifeng Hot Spring. Furthermore, it was found that genus Flavobacterium was susceptible to environmental change with abundance of 11 ~ 2825 times higher in OWD than the other two groups. Compared bacteria from the OWD group with that from 14 hot springs in six countries, total 94 unique genera bacteria were found out from the Xifeng Hot Spring including four thiometabolism-related bacteria (Propionispira, Desulforegula, Desulfobacter and Desulfococcus) and the thermophilic bacterium (Symbiobacterium). Using microbial culturing and isolation technology, sixteen strains were isolated from the water samples of three sites. The diversity of microbiota was abundant and variable along with the niche changed in conditions and surroundings. It indicated that numbers of valuable bacteria resources could be explored from the special surroundings of Xifeng Hot Spring.