The biofilm characteristics (population dynamics and biofilm composition) in a biological filter for the removal of iron, manganese and ammonium were studied in a drinking water treatment plant. The objective was to examine the spatial distribution and biological composition of active biomass that grows in a biological filter and to verify the effect of the backwashing on the quantity of fixed biomass and on the density and activity of the biological population. Heterotrophic microorganisms activity was higher in the upper layer of the filter. Nitrifying microorganisms colonized the biofilter in a stratified manner and their activity was higher in the second layer of the filter. A total of 14 species of ciliated protozoa and 7 species of filamentous microorganisms were found in the biofilters. Ciliates were concentrated in the filterbed layer in which the heterotrophic activity was higher. The grazing activity of ciliates on heterotrophic bacteria reduced the competition pressure on nitrifying microorganisms, supporting their growth and thus raising the ammonium removal efficiency. In general, filamentous microorganisms appeared to be indifferent to operating changes in the plant such as backwashing and filtering cycles. Crenothrix was the prevalent filamentous microorganism in terms of both frequency and abundance; it was found prevalently in the first layer where the oxidisation of iron and manganese occurred.