Understanding the influences of biotic and abiotic factors on microbial community structure and methanogenesis are important for its engineering and ecological significance. In this study, four biogas digesters were supplied with the same inoculum and feeding sludge but operated at different sludge retention time (7 to 16 days) and organic loading rates for 90 days to determine the relative influence of biotic and environmental factors on the microbial community assembly and methanogenic performance. Despite different operational parameters, all digester communities were dominated by Bacteroidales, Clostridiales, and Thermotogales and followed the same trend of population dynamics over time. Network and multivariate analyses suggest that deterministic factors, including microbial competition (involving Bacteroidales spp.), niche differentiation (e.g., within Clostridiales spp.), and periodic microbial immigration (from feed sludge), are the key drivers of microbial community assembly and dynamics. A yet-to-be-cultured phylotype of Bacteroidales (GenBank ID: GU389558.1) is implicated as a strong competitor for carbohydrates. Moreover, biogas-producing rate and methane content were significantly related with the abundances of functional populations rather than any operational or physicochemical parameter, revealing microbiological mediation of methanogenesis. Combined, this study enriches our understandings of biological and environmental drivers of microbial community assembly and performance in anaerobic digesters.