In this study, temporal impacts of thermal, alkaline/acid and thermal-alkaline sludge pre-treatments on digestion performance and microbiome were investigated and compared in methanogenic sludge digesters. Results showed that thermal and alkaline/acid pre-treatments were efficient in releasing intracellular and EPS organic matter, respectively. The thermal-alkaline pre-treatment showed synergistic impacts of both thermal and alkaline/acid pre-treatments by releasing the major portion of sludge organic matter from solid- to liquid-phase, which result in 60–65% organic carbon removal in subsequent sludge digestion and further optimizing digestion temperature had negligible improvement. The 16S rRNA gene-based analyses suggested that organic matter released from sludge pre-treatments is a major deterministic parameter in shaping sludge microbiome. Pre-treatment specific lineages were identified in different sludge digesters, whereas several taxa were identified as common functionally active populations in sludge digestion. This study provided mechanistic insights into impacts of pre-treatments on digestion performance and microbiome in methanogenic sludge digesters.