A substantial portion of the carbon (C) fixed by the trees is allocated belowground to ectomycorrhizal (EM) symbionts, but this fraction usually declines after fertilization. The aim of the present study was to estimate the effect of optimal fertilization (including all the necessary nutrients) on the growth of EM fungi in young Norway spruce forests over a three year period. In addition, the amount of carbon sequestered by EM mycelia was estimated using a method based on the difference in δ¹³C between C₃ and C₄ plants. Sand-filled ingrowth mesh bags were used to estimate EM growth, and similar bags amended with compost made from maize leaves (a C₄ plant) were used to estimate C sequestration. Fertilizers had been applied either every year or every second year since 2002 and the estimates of EM growth started in 2007. The application of fertilizer reduced EM growth to between 0% and 40% of the growth in the control plots at one site (Ebbegärde), while no significant effect was found at the other three sites studied. The effect of the fertilizer was similar in sand-filled and maize-compost-amended mesh bags, but the total production of EM fungi was 3–4 times higher in maize-compost-amended mesh bags. The fertilizer tended to reduce EM growth more when applied every year than when applied every second year. The amount of C sequestered in maize-compost-amended mesh bags collected from unfertilized treatments was estimated to be between 0.2 and 0.7 mg C g sand⁻¹ at Ebbegärde and between 0.2 and 0.5 mg C g sand⁻¹ at Grängshammar. This corresponds to between 300 and 1100 kg C per ha, assuming a similar production in the soil as in the mesh bags. Fertilization at the Ebbegärde site reduced carbon sequestration, which confirmed the results based on estimates of fungal growth (ergosterol levels). A correlation was found between fungal biomass and δ¹³C in mesh bags amended with maize compost. Based on this, it was estimated that a fungal production of 1 μg ergosterol corresponded to 0.33 mg of sequestered carbon. In conclusion, the effect of the fertilizer on EM growth seemed to be dependent on the effect of the fertilizer on tree growth. Thus, at Ebbegärde, were tree growth was less stimulated by the fertilizer, EM growth was reduced upon fertilization. At other sites, where tree growth was more stimulated, the fertilizer did not influence EM growth. The large amounts of carbon sequestered during the experiment may be a result of fungal residues remaining in the soil after the death of the hyphae.