Recently, thermophilic microaerobic pretreatment (TMP) showed to be an efficient pretreatment method for anaerobic digestion (AD). During TMP, bacteria affiliated to phylum Firmicutes played an important role. The microbial community structure of inoculum might directly affect the performance of TMP. In this study, three different mixed microfloras [biogas slurry (BS), activated sludge (AS), and effluent from retted corn straw (ER)] were selected to explore inocula impact on the performance of TMP. Results showed that the TMP process could obviously improve the startup performance and methane yield of AD. Despite the inocula during TMP being different when the oxygen load during TMP was 5 mL/g of VSsubstrate, the methane yields reached a maximum. The maximum methane yield was obtained when using ER as inoculum during TMP, Which was 5.2 and 5.8% higher than those of samples using BS and AS as inocula, respectively. Accordingly, the relative abundance of phylum Firmicutes in ER was also highest, which. was 77.4 and 459.5% higher than those of BS and AS, respectively. When using TMP as the pretreatment method, the inoculum selection during the TMP process is important, and the higher abundance of bacteria affiliated to phylum Firmicutes could improve the performance of TMP.