To provide the first step towards a microbial ecology-based understanding of irreversible membrane biofouling, four laboratory-scale membrane bioreactors (MBRs) were operated to investigate the identity of bacterial populations highly correlated with irreversible membrane biofouling. The conventional MBR was divided into two separate experimental units. Unit one consisted of four suspended-growth, activated sludge, sequencing batch bioreactors treating a synthetic paper mill wastewater. Unit two consisted of a microfiltration membrane cell. Amplified ribosomal deoxyribonucleic acid restriction analysis (ARDRA) was used to compare the predominant bacterial populations in samples of mixed liquor and irreversibly bound to the membrane surface. The results of ARDRA showed a significant difference between the planktonic and sessile bacterial communities suggesting that irreversible biofouling of microfiltration membranes may be more highly correlated to specific bacterial populations rather than the total, bulk concentration of biomass. A custom-built mini-flow cell and light microscopy were used to visualise the early formation of biofilms by two pure cultures (Escherichia coli and Acinetobacter calcoaceticus) on membrane surfaces. The results confirmed that A. calcoaceticus was able to enhance the initiation of biofilm formation on microfiltration membranes.