Dark fermentative biohydrogen production in a thermophilic, xylose-fed (50 mM) fluidised bed reactor (FBR) was evaluated in the temperature range 55–70 °C with 5-degree increments and compared with a mesophilic FBR operated constantly at 37 °C. A significantly higher (p = 0.05) H2 yield was obtained in the thermophilic FBR, which stabilised at about 1.2 mol H2 mol−1 xylose (36% of the theoretical maximum) at 55 and 70 °C, and at 0.8 mol H2 mol−1 xylose at 60 and 65 °C, compared to the mesophilic FBR (0.5 mol H2 mol−1 xylose). High-throughput sequencing of the reverse-transcribed 16S rRNA, done for the first time on biohydrogen producing reactors, indicated that Thermoanaerobacterium was the prevalent active microorganism in the thermophilic FBR, regardless of the operating temperature. The active microbial community in the mesophilic FBR was mainly composed of Clostridium and Ruminiclostridium at 37°C. Thermophilic dark fermentation was shown to be suitable for treatment of high temperature, xylose-containing wastewaters, as it resulted in a higher energy output compared to the mesophilic counterpart.