We analysed prokaryotic and eukaryotic communities across the seawater pre-treatment system of Penneshaw (Kangaroo Island, South Australia) desalination plant, using 16S and 18S rRNA gene sequencing. The richness of operational taxonomic units increased downstream of the pre-treatmentsystem (reverse osmosis feedwater) compared to raw seawater for Archaea, while it decreased for bacteria and protists. Overall, the reverse osmosis feedwater was found to be enriched in ammonia-oxidising bacteria and Archaea compared to raw seawater, and also contained greater proportions of taxa typically observed in aquatic biofilms and/or within other water treatment systems. Although the microbial load was reduced by the pre-treatment system, the increase in proportion of biofilm-associated microbes suggests the presence of active microbial communities within multimedia filters and other parts of the pre-treatment system that might increase biofouling risks.