Here we present a new approach to quantify food-web interactions within semi-natural biofilms by combining the establishment of biofilms from natural rivers in flow cells with video microscopy. In a first application of this approach, we focused on the surface-gliding heterotrophic flagellates (HF) Neobodo designis, Rhynchomonas nasuta and Planomonas sp. It was shown that the three HF generally ingested single biofilm-associated bacteria whereas bacteria within microcolonies were attacked but not ingested. However, grazing strategies differed considerably. While the kinetoplastids N. designis and R. nasuta displayed long search and short handling times, Planomonas sp. showed the opposite grazing characteristics. The latter behaviour resulted in a high relative predation success of 80% (precent of attacked prey ingested), whereas the relative predation success of the two kinetoplastids was only 20%. However, the two contrasting strategies resulted in similar ingestion rates for Planomonas sp. and N. designis of 0.5 to 0.6 ingestions flagellates−1 minute−1, respectively. Our results showed distinct differences in the feeding behaviour of three flagellates having similar life forms and provide direct evidence that microcolony formation in biofilms protects bacteria from grazing by HF in situ.