Dual media filter (DMF) is being used as a primary pretreatment to remove particulate foulants at seawater desalination plants. However, many plants experience organic and biological fouling. The first part of this paper focuses on the monitoring of organic and biological foulants using advanced analytical techniques to optimize functioning of DMF at Perth Seawater Desalination Plant (PSDP) in Western Australia. In addition, microbial community analysis in DMF filtered seawater, and on DMF media (DMF-M) and cartridge filter (CF) was conducted using terminal restriction fragment length polymorphism (T-RFLP) and 454-pyrosequencing. In the full-scale DMF system, the bacterial community structure was clustered along with the filtration time and sampling positions. For the DMF effluent samples, the bacterial community structure significantly shifted after 4 h of filtration time, which corresponded with the permeability reduction trend. The dominant bacterial communities in the DMF effluent were OTU 13 (Phaeobacter) and OTU 19 (Oceaniserpentilla). The different biofilm-forming bacteria communities were found in the biofilm samples on DMF-M and CF. In the second part of the study, semi-pilot scale DMF columns were operated on-site under same operating conditions used in PSDP. It demonstrated the advantage of operating DMF at the biofiltration mode for improving the reduction of biofoulants. © 2016 Elsevier B.V.