Resumo. The secondary metabolites extracted from forest species known as micossimbiontes can stimulate the growth of in vitro ectomycorrhizal isolates. We determined the effect of concentration of Eucalyptus grandis essential oil in stimulating growth of the ectomycorrhizal Pisolithus sp. (UFSC Pt 24 and UFSC Pt 188), Pisolithus microcarpus (UFSC Pt 116) Chondrogaster angustiporus (UFSC Ch 163), Scleroderma citrinum (UFSC Sc 124) and Suillus sp. (UFSM RA RA UFSM 2.2 and 2.8) in liquid culture medium. After a period of 25 days of incubation, we evaluated the morphology and growth of the isolates. The addition of essential oil at concentrations from 20 to 30 µL L-1 promoted mycelial growth in vitro in isolated Pt 24 UFSC, UFSC Pt 116, Ch 163 UFSC, UFSC Sc 124, UFSM RA RA UFSM 2.2 and 2.8. The addition of essential oil at a concentration of 20 µL L-1 resulted in an increase in the diameter and branching hyphae of isolates UFSC Pt 116 and Pt 24 UFSC. The use of essential oil in the preparation of culture media for the growth of ectomycorrhizal isolates has shown to be efficient and to increase the mycelial mass of the fungus.