High-loaded membrane bioreactors (HL-MBRs), i.e., bioreactors equipped with a membrane for biomass retention and operated at extremely short sludge and hydraulic retention times, can concentrate sewage organic matter to facilitate subsequent energy and chemical recovery from these organics. Bioflocculation, accomplished by microorganisms that produce extracellular polymers, is a very important mechanism in these reactors. Bacterial diversity of the sludge and supernatant fraction of HL-MBRs operated at very short sludge retention times (0.125, 0.5, and 1 day) were determined using a PCR-denaturing gradient gel electrophoresis (DGGE) and clone library approach and compared to the diversity in sewage. Already at a sludge retention time (SRT) of 0.125 day, a distinct bacterial community developed compared to the community in sewage. Bioflocculation, however, was low and the majority of the bacteria, especially Arcobacter, were present in the supernatant fraction. Upon increasing SRT from 0.125 to 1 day, a much stronger bioflocculation was accompanied by an increased abundance of Bacteroidetes in the (solid) sludge fraction: 27.5 % at an SRT of 0.5 day and 46.4 % at an SRT of 1 day. Furthermore, cluster analysis of DGGE profiles revealed that the bacterial community structure in the sludge was different from that in the supernatant. To localize specific bacterial classes in the sludge flocs, fluorescence in situ hybridization (FISH) was carried out with three different bacterial probes. This showed that Betaproteobacteria formed clusters in the sludge flocs whereas Alphaproteobacteria and Gammaproteobacteria were mainly present as single cells.