Ectomycorrhizal (EM) fungi form symbioses with dominant tree families in boreal, temperate and tropical ecosystems and are important drivers of ecosystem function. EM fungal hyphae extend over a large area making them susceptible to enhanced radiation levels from naturally occurring or anthropogenically originating radioisotopes in the rhizosphere. In this study, the in-vitro effects of ionizing radiation on the growth and biomass of EM fungi Suillus luteus, S. bovinus and Rhizopogon luteolus were investigated. EM fungal cultures were exposed to gamma radiation from a 137Cs source for 137 h in darkness at 21 °C at dose rates of 404, 108.5 and 54.9 mGy h−1 resulting in total absorbed doses of 55.21, 14.82 and 7.50 Gy respectively. Cultures grown in the dark at 21 °C but not exposed to the 137Cs source served as the control. Our results show that EM fungi vary in their sensitivity to ionizing radiation. EM fungi used in this study produced melanin and reactive oxygen species scavenging enzymes such as catalase and superoxide dismutase as a response to ionizing radiation.