The pretreatment of lignocellulosic substrates with cattle rumen fluid was successfully developed to increase methane production. In the present study, a 16S rRNA gene-targeted amplicon sequencing approach using the MiSeq platform was applied to elucidate the effects of the rumen fluid treatment on the microbial community structure in laboratory-scale batch methane fermenters. Methane production in fermenters fed rumen fluid-treated rapeseed (2,077.3 mL CH4 reactor-1 for a 6-h treatment) was markedly higher than that in fermenters fed untreated rapeseed (1,325.8 mL CH4 reactor-1). Microbial community profiling showed that the relative abundance of known lignocellulose-degrading bacteria corresponded to lignocellulose-degrading enzymatic activities. Some dominant indigenous cellulolytic and hemicellulolytic bacteria in seed sludge (e.g., Cellulosilyticum lentocellum and Ruminococcus flavefaciens) and rumen fluid (e.g., Butyrivibrio fibrisolvens and Prevotella ruminicola) became undetectable or markedly decreased in abundance in the fermenters fed rumen fluid-treated rapeseed, whereas some bacteria derived from seed sludge (e.g., Ruminofilibacter xylanolyticum) and rumen fluid (e.g., R. albus) remained detectable until the completion of methane production. Thus, several lignocellulose-degrading bacteria associated with rumen fluid proliferated in the fermenters, and may play an important role in the degradation of lignocellulosic compounds in the fermenter.