Dense microbial mats and streamers of various colors developed in an alkaline-hot spring water at 48–76°C and ~0.077 mM sulfide in Nakabusa, Japan. The microbial community structures with a thermal gradient were compared by denaturing gradient gel electrophoresis (DGGE) analysis of the PCR-amplified 16S ribosomal RNA gene fragments. The sequence analysis revealed that a predominant cyanobacterial DGGE band phylogenetically related to Synechococcus elongatus was detected only from green mats at 48°C. Four DGGE bands were detected commonly from green mats at 48°C, orange mats at 58°C and brown mats at 60°C. The sequence analysis revealed that these were phylogenetically related to Chloroflexaceae group, Rhodothermus group, a candidate division OP10, and an unclassified bacterium. On the other hand, Aquificae-, Thermodesulfobacteria-, Thermus group-, and Crenarchaeota-like sequences were detected as a predominant component of DGGE profiling from the streamers only at temperatures over 66°C, but no phototrophic bacterial bands were detected. Thus, the microbial community structure above 60°C was drastically different from that at the lower temperatures. After the addition of hydrogen into in vitro gray streamers with in situ spring water, sulfide production markedly occurred in the presence of ambient sulfate at 66°C. This result suggests that in situ sulfide is partly produced by Thermodesulfobacteria-like sulfate-reducing bacteria in the streamers.