The composition and seasonal dynamics of biofilm-associated eukaryotic communities were analysed at the metre and kilometre scale along a salinity gradient in the Westerschelde estuary (The Netherlands), using microscopy and a genetic fingerprinting technique (PCR-DGGE). Microphytobenthic biomass, measured as chlorophyll a (chl a), varied seasonally over 2 orders of magnitude, being highest in spring. Communities were dominated by epipelic diatoms, in particular by members of the genus Navicula. In spring, a few smaller epipelic diatom species dominated during biomass peaks, while during the rest of the year, communities were more diverse and were characterised by larger species. The microphytobenthic community collapsed when grazers appeared, which happened concomitantly with a rise in temperature. Spring biomass development was associated with marked changes in porewater nutrient concentrations, especially towards the estuary mouth. In the DGGE data, diatoms, ciliates, amoebae, copepods, nematodes, annelids and platyhelminthes were detected. Ordination analysis of the species counts and DGGE data were largely congruent and indicated that on the scale of the whole estuary (i.e. km scale), taxonomic turnover in microphytobenthos composition was mainly associated with the salinity gradient. At smaller spatial scales, the position of sampling localities along the tidal exposure gradient appeared to be the main determinant of species turnover, in particular in the brackish reaches of the estuary.