Abstract Aspen and balsam poplar seedlings were inoculated with six species of ectomycorrhizal fungi (Hebeloma longicaudum, Laccaria bicolor, Paxillus involutus, Pisolithus tinctorius, Rhizopogon vinicolor, and Suillus tomentosus), one species of endomycorrhizal fungus (Glomus intraradices), two species of bacteria (Agrobacterium sp. and Burkholderia cepacia), treated with a growth hormone (SR3), and co-inoculated with a combination of Paxillus and Burkholderia. The seedlings were grown in a greenhouse under three different fertility regimes. Bacterial inoculation alone did not affect seedling growth and nutrition as observed when co-inoculated with ectomycorrhizal fungus. The biomass and root collar diameter of aspen and balsam poplar were significantly increased when adequate mycorrhizas are formed and more prominent when co-inoculated with P. involutus and B. cepacia and grown at the 67% fertilizer level. Except for R. vinicolor and S. tomentosus, the other four species of ectomycorrhizal fungi and G. intraradices formed symbiotic associations with both plant species. Both ectomycorrhizal and endomycorrhizal colonization were observed at all fertilizer levels and fertilizer applications did not affect the colonization rates. Nitrogen and phosphorus concentrations were significantly improved in both aspen and balsam poplar compared with control only when co-inoculated with P. involutus and B. cepacia. However, plant net nitrogen uptake (content) increased significantly in all successful inoculation treatments and co-inoculated treatment when compared with control. These results hold promise for incorporation of inoculation of Populus sp. with appropriate mycorrhizal fungi and selected bacteria into commercial nursery system to improve the establishment of Populus in various sites.