Although microorganisms are the primary drivers of biogeochemical cycles, the structure and functioning of microbial food webs is poorly studied. This is the case in Sphagnum-peatlands, where microbial communities play a key role in the global carbon cycle. Here, we explored the structure of the microbial food web from a Sphagnum-peatland by analysing (1) the density and biomass of different microbial functional groups, (2) natural stable isotope (δ13C and δ15N) signatures of key microbial consumers (testate amoebae), and (3) the digestive vacuole contents of Hyalosphenia papilio, the dominant testate amoeba species in our system. Our results showed that the feeding type of testate amoeba species (bacterivory, algivory or both) translates into their trophic position as assessed by isotopic signatures. Our study further demonstrates, for Hyalosphenia papilio, the energetic benefits of mixotrophy when the density of its preferential prey is low. Overall, our results show that testate amoebae occupy different trophic levels within the microbial food web, depending on their feeding behaviour, the density of their food resources and their metabolism (i.e. mixotrophy vs. heterotrophy). Combined analyses of predation, community structure and stable isotopes now allow the structure of microbial food webs to be more completely described, which should lead to improved models of microbial community function.