ABSTRACT: We analysed prokaryotic and eukaryotic communities across the seawater pre-treatment system of a desalination plant, using 16S and 18S rRNA gene sequencing. The richness of operational taxonomic units increased downstream of the pre-treatment system (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 is 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.