Ectomycorrhizal fungi (ECMF) are an important biotic factor in the survival of conifer seedlings under stressful conditions and therefore have the potential to facilitate conifer establishment into alpine and tundra habitats. In order to assess patterns of ectomycorrhizal availability and community structure above treeline, we conducted soil bioassays in which Picea mariana (black spruce) seedlings were grown in field-collected soils under controlled conditions. Soils were collected from distinct alpine habitats, each dominated by a different ectomycorrhizal host shrub: Betula glandulosa, Arctostaphylos alpina or Salix herbacaea. Within each habitat, half of the soils collected contained roots of ectomycorrhizal shrubs (host+) and the other half were free of host plants (host−). Forest and glacial moraine soils were also included for comparison. Fungi forming ectomycorrhizae during the bioassays were identified by DNA sequencing. Our results indicate that ECMF capable of colonizing black spruce are widespread above the current tree line in Eastern Labrador and that the level of available inoculum has a significant influence on the growth of seedlings under controlled conditions. Many of the host− soils possessed appreciable levels of ectomycorrhizal inoculum, likely in the form of spore banks. Inoculum levels in these soils may be influenced by spore production from neighboring soils where ectomycorrhizal shrubs are present. Under predicted temperature increases, ectomycorrhizal inoculum in soils with host shrubs as well as in nearby soils without host shrubs have the potential to facilitate conifer establishment above the present tree line.