Using a culture-independent method that combines CARD-FISH, qPCR and 16S rDNA, we investigated the abundance, community structure and diversity of microbes along a steep thermal gradient (50–90 °C) in the Tengchong Geothermal Field. We found that Bacteria and Archaea abundance changed markedly with temperature changes and that the number of cells was lowest at high temperatures (90.8 °C). Under low-temperature conditions (52.3–74.6 °C), the microbial communities were dominated by Bacteria, which accounted for 60–80% of the total number of cells. At 74.6 °C, Archaea were dominant and at 90.8 °C, they accounted for more than 90% of the total number of cells. Additionally, the microbial communities at high temperatures (74.6–90.8 °C) were substantially simpler than those at the low-temperature sites. Only a few genera (e.g., bacterial Caldisericum, Thermotoga and Thermoanaerobacter, archaeal Vulcanisaeta and Hyperthermus) often dominated in high-temperature environments. Additionally, a positive correlation between Ammonia-Oxidizing Archaea (AOA) activity and temperature was detected. AOA activity increased from 17 to 52 pmol of NO₂⁻ per cell d⁻¹ with a temperature change from 50 to 70 °C.