The prevailing N saturation paradigm still considers microbial N immobilization as a less important process of ecosystem N retention. On the contrary, we hypothesize that it can even be a primary process affecting N leaching from N saturated soils. We studied N transformations in soils of acidified near-natural and primeval forests in the Bohemian Forest (watersheds of Plešné and Čertovo Lakes, Czech Republic) and Pop Ivan Massif (Ukraine). Organic soils were sampled from similar conditions (1100–1500 m a.s.l., precipitation 1400–1800 mm, acidic bedrock, forest dominated by Picea abies) and had similar chemical properties (pHKCl 2.5–3.2, BS ∼45%, Alex ∼40 meq kg−1). However, the Ukrainian soil had lower soil C/N ratio (24 vs. 30) and C availability (water soluble C and C/N ratio of 65 vs. ∼114–163 μmol g−1 and 6 vs. 21–24, respectively) than the other soils. We ran laboratory experiments in which mixtures of different N sources (N–NH4, N–NO3 and glycine) were added to the soil with only one source 15N-labelled. We followed 15N partitioning within soil N pools and analysed the composition of the microbial community (16SrDNA-DGGE fingerprint of bacteria, ergosterol analyses, qPCR of fungal 18S rDNA gene). The microbial N pool was always three to five times higher than the total soluble N pool. We found rapid (15 min) and simultaneous immobilization of all added N forms into the microbial biomass with clear preferences for organic N over inorganic sources. The total N flux to the microbial pool always exceeded N flux into mineral N pools. The pattern of N transformation in the C limited soil was different from the other soils. The microbial pool and N flux into it were smaller compared to the mineral N pools and fluxes. The contribution of N–NO3 to microbial immobilization was negligible, while nitrification was almost equal to N mineralization. Total N flux through soluble N pools was greater than total N flux to insoluble pools (residual and microbial N); this was accompanied by lower microbial N uptake efficiency and shorter residence time of N in microbial pool than in soils with higher C availability. The composition of bacterial community was related to DOC content and C and N in microbial biomass. In soils with higher fungal abundance, more glycine was immobilized regardless of soil C availability, but with higher deamination (∼50 vs. 20%) and subsequent release of N–NH4 back to the soil. Our study emphasized the role of microbial N immobilization in preventing N–NO3 loss from N saturated ecosystems as a function of C availability. Nitrification was favoured when enough N–NH4 was available in the C limited soil. The discharged N–NO3 was not immobilized by the microbes and could be, if not immobilized by plants, leached out. C limitation plays an important role in the susceptibility of ecosystems to N leaching and could partially explain the observed differences in some N-saturated ecosystems.