In present study, the impact of air and soil temperature and CO2 concentration on beech seedlings and on ectomycorrhizal fungi (ECM) was studied. The experiment was conducted on one year old beech seedlings (Fagus sylvatica L.) planted into rhizotrons and exposed to different temperature: cooled chamber (15-20 °C) without cooling of roots (CR-) and with additional cooling of roots for 4-5 °C (CR+), greenhouse (GH) with elevated temperatures and outside (OUT).. During the experiment nondestructive analyzes were made, while in the end of tree-year experiment destructive analyzes of whole seedlings was performed. The highest proportion of fine roots, more than 98% and the lowest average root diameter were observed in GH. In CR+, CR- and GH between 10,3% and 11,4% of mycorrhizal roots were observed, while OUT were for half less mycorrhizal roots (5%). From the obtained measurements different growth parameters were recorderd. The significant impact (p<0,05) of root temperature on specific root length were observed and significant reduction (p<0,001) of specific leaf area and leaf area ratio as a consequence of elevated CO2. Mycorrhizal fungi were identified using anatomical-morphological, molecular and phylogenetic analyzes. 114 ECM types were identified. Two-photon confocal microscope and ELF-97% fluorescent dye were used for establishing the activity and occurrence of surface-bound phosphatase activity in different exploration types (CT ET, SD-ET, MD-ET, LD-ET) of ECM. Occurrence of surface bound acid phosphatase was significant (p<0,001) between ET, while temperature had no significant effect. The %15N in %13C analyzes in different parts of plant tissues, mycorrhizal fungi and soil were used to establish nitrogen and carbon flow between fungi and host plant and the influence of environmental conditions on it. Mycorrhizal fungi are very diverse group influenced by temperature and elevated CO2 concentrations. Enzymatic activity of exploration types depended on environmental factors and of function of ectomycorrhizal fungi that they had in the system.