There is debate about whether results from mesocosm studies should be extrapolated to natural ecosystems. Critics argue that the abiotic and biotic characteristics of mesocosms do not represent the in situ conditions of the habitats in which they are designed to mimic. We addressed this idea by deploying mesocosms in two lakes of varying trophic status (mesotrophic and eutrophic). Water chemistry was relatively similar between the mesocosms and the respective lakes. In contrast, biological variables including most phytoplankton, nanoflagellates, ciliates, and zooplankton were lower in the mesocosms than they were in the respective lakes. There was also a shift in zooplankton composition such that mesocosm assemblages diverged from pelagic to littoral communities. While our results show that freshwater planktonic organisms can vary considerably between mesocosms and in situ habitats, we also suggest that factors such as isolation from lake sediments, the lack of predators, and the temporal scale at which mesocosms are filled and sampled should be considered when designing and interpreting mesocosm studies. Nevertheless, mesocosms are an important tool for developing and testing general ecological hypotheses that cannot be achieved using other approaches alone.