This study examined the composition and structure of nitrifying biofilms sampled from a high-rate nitrifying trickling filter which was designed to pre-treat raw surface water for potable supply. The filter was operated under a range of feed water ammonia and organic carbon concentrations that mimicked the raw water quality of poorly protected catchments. The biofilm structure was examined using a combination of fluorescence in situ hybridisation and scanning electron microscopy. Biopolymers (carbohydrate and protein) were also measured. When the filter was operated under low organic loads, nitrifiers were abundant, representing the majority of microorganisms present. Uniquely, the study identified not only Nitrospira but also the less common Nitrobacter. Small increases in organic carbon promoted the rapid growth of filamentous heterotrophs, as well as the production of large amounts of polysaccharide. Stratification of nitrifiers and heterotrophs, and high polysaccharide were observed at all filter bed depths, which coincided with the impediment of nitrification throughout most of the filter bed. Observations presented here specifically linked biofilm structure with filter functionality, physically validating previous empirical modelling hypotheses regarding competitive interactions between autotrophic and heterotrophic bacteria in biofilms.