Climate change is likely to cause increased tree recruitment on open peatlands but we currently have little idea what consequences this vegetation change may have below-ground. Here we use transects across forested to open bog ecotones at three Russian peatland complexes to assess potential changes in the most abundant group of peatland protists – the testate amoebae. We show that the testate amoeba communities of forested and open bog are markedly different with a very abrupt boundary at, or near, the vegetation ecotone. Changes along our transects suggest that tree encroachment may reduce the trophic level of testate amoeba communities and reduce the contribution of mixotrophic testate amoebae to primary production. Our study strongly suggests that increased tree recruitment on open peatlands will have important consequences for both microbial biodiversity and microbially-mediated ecosystem processes.