Deciduous forests may respond differently from coniferous forests to the anthropogenic deposition of nitrogen (N). Since fungi, especially ectomycorrhizal (EM) fungi, are known to be negatively affected by N deposition, the effects of N deposition on the soil microbial community, total fungal biomass and mycelial growth of EM fungi were studied in oak-dominated deciduous forests along a nitrogen deposition gradient in southern Sweden. In-growth mesh bags were used to estimate the production of mycelia by EM fungi in 19 oak stands in the N deposition gradient, and the results were compared with nitrate leaching data obtained previously. Soil samples from 154 oak forest sites were analysed regarding the content of phospholipid fatty acids (PLFAs). Thirty PLFAs associated with microbes were analysed and the PLFA 18:2 omega 6,9 was used as an indicator to estimate the total fungal biomass. Higher N deposition (20 kg N ha(-1) y(-1) compared with 10 kg N ha(-1) y(-1)) tended to reduce EM mycelial growth. The total soil fungal biomass was not affected by N deposition or soil pH, while the PLFA 16:1 omega 5, a biomarker for arbuscular mycorrhizal (AM) fungi, was negatively affected by N deposition, but also positively correlated to soil pH. Other PLFAs positively affected by soil pH were, e.g., i14:0, a15:0, 16:1 omega 9, a17:0 and 18:1 omega 7, while some were negatively affected by pH, such as i15:0, 16:1 omega 7t, 10Me17:0 and cy 19:0. In addition, N deposition had an effect on the PLFAs 16: 1 omega 7c and 16:1 omega 9 (negatively) and cy 19:0 (positively). The production of EM mycelia is probably more sensitive to N deposition than total fungal biomass according to the fungal biomarker PLFA 18:2 omega 6,9. Low amounts of EM mycelia covaried with increased nitrate leaching, suggesting that EM mycelia possibly play an important role in forest soil N retention at increased N input.