Autotrophic nitrogen removal technologies have proliferated through the last decade. Among these, a promising one is the membrane bioreactor (MBR) Anammox, which can achieve very high solids retention time and therefore sets a proper environment for the cultivation of anammox bacteria. In this sense, the MBR Anammox is an efficient technology for the treatment of effluents with low organic carbon and high ammonium concentrations once it has been treated under partial nitrification systems. A lab‐scale MBR Anammox bioreactor has been built at the Technological University of Delft, The Netherlands and has been proven for efficient nitrogen removal and efficient cultivation of anammox bacteria. In this study, next‐generation sequencing techniques have been used for the investigation of the bacterial communities of this MBR Anammox for the first time ever. A strong domination of Candidatus Brocadia bacterium and also the presence of a myriad of other microorganisms that have adapted to this environment were detected, suggesting that the MBR Anammox bioreactor might have a more complex microbial ecosystem that it has been thought. Among these, nitrate‐reducing heterotrophs and primary producers, among others, were identified. Definition of the ecological roles of the OTUs identified through metagenomic analysis was discussed. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:186–193, 2015