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Temperature plays a significant role in anaerobic digestion (AD) as it affects the microbial communities and ultimately controls the efficiency of the process. Few studies have looked at temperature-adjusted AD, but it is unclear how the temperature shifts affect biogas production and the dynamics of microorganisms involved in methanogenesis. This study tested two temperature shift scenarios in fed-batch mode using anaerobically digested sewage sludge and glucose-based substrate. The first scenario was acclimatized to upshifting temperatures from 42 °C to 48 °C while the second was acclimatized to downshifting temperatures from 55 °C to 45 °C. Both temperature shift scenarios resulted in a decrease in biogas production, especially at 45 °C. The upshifted scenario experienced a maximum decrease of 83%, and the downshifted scenario experienced a 16–33% decrease in methane production. Next-generation 16S rRNA sequencing revealed the domination of <i>Methanoculleus</i> in the upshifted scenario. However, a low correlation between the number of <i>Methanoculleus</i> and the other hydrogenotrophic methanogens to biogas production indicates inhibition in the hydrogenotrophic pathway. The downshifted scenario showed better biogas production due to the substantial domination of acetoclastic <i>Methanosaeta</i> and the low abundance of sulfate-reducing bacteria. Hence, the temperature shift affects the microbial communities, significantly affecting biogas production performance.