Aims The purpose of this study was to apply cDNA approach for the characterization of active prokaryotic community to understand microbial scenarios and performance of an AnSBR digester fed with acid hydrolysates of Agave tequilana var. azul bagasse (ATAB). Methods and Results The digester was implemented for methane production under organic loading rate (OLR) disturbances to correlate physicochemical variables with changes in abundance, diversity and population dynamics of active Bacteria and Archaea by principal components analysis (PCA). Results indicated that methane yield increased as well as active syntrophic relationships for interspecies hydrogen/formate (Anaerolinaceae-Methanobacterium beijingense) and acetate (Anaerolinaceae-Methanosaeta concilii) transfers at 8 g-COD l(-1) day(-1). However, methane yield was negatively affected at 16 g-COD l(-1) day(-1) due to the competition for acetate by active Desulfovibrio marrakechensis and volatile fatty acids inhibition. Conclusions Microbial scenarios obtained by PCA correlations indicated that methane production from acid hydrolysates of ATAB was feasible at 8 g-COD l(-1) day(-1). The digester operation at higher OLR only favoured methanogenesis by the hydrogenotrophic pathway. Significance and Impact of the Study Only cDNA analysis showed Archaea population dynamics, exhibiting high correlation with physicochemical variables towards the understanding of the methanogenic digester performance during OLR disturbances.