Thermophile pretreatment of activated sludge greatly improves the biodegradability of sludge, but whether the pretreated products are suitable for the electricity generation of microbial fuel cells (MFCs) is still little known. In this study, municipal activated sludge pretreated by a thermophilic bacterium and heating, respectively, was separately fed into the MFCs. The performance of MFCs was examined and changes of anodic microbial communities were investigated with scanning electron microscopy and 16S rRNA gene high-throughput sequencing on the Illumina Miseq platform. The results showed that MFCs fed with heating-pretreated sludge performed preferably and the power density reached 0.91–2.86 W/m3. MFC anodes were covered with considerable Geobacter spp. However, the bioaugmentation of sludge with the thermophile was not able to support a high potential output although the pretreatment significantly increased the soluble chemical oxygen demand. The maximum power density approached 0.20 W/m3 even when the anolyte was regularly changed. It was observed that amending pH did not improve the performance of MFC. Investigation on this anodic microbial community found that the relative abundance of Lactobacillus spp. exceeded 91%. Consequently, the thermophile-pretreated products stimulated the growth of non-exoelectrogens and finally the niches of anodic biofilm were completely occupied by Lactobacillus spp.