The interactions between dissolved organic matter (DOM) and mineral particles are critical for the stabilization of soil organic matter (SOM) in terrestrial ecosystems. The processing of DOM by ectomycorrhizal fungi contributes to the formation of mineral-stabilized SOM by two contrasting pathways: the extracellular transformation of DOM (ex vivo pathway) and the secretion of mineral-surface-reactive metabolites (in vivo pathway). In this study, we examined how changes in nitrogen (N) availability affected the formation of mineral-associated carbon (C) from these two pathways. DOM was extracted from forest soils. The processing of this DOM by the ectomycorrhizal fungus Paxillus involutus was examined in laboratory-scale studies with different levels of ammonium. At low levels of ammonium (i.e., under N-limited conditions), the DOM components were slightly oxidized, and fungal C metabolites with iron-reducing activity were secreted. Ammonium amendments decreased the amount of C metabolites, and no additional oxidation of the organic matter was detected. In contrast, the hydrolytic activity and the secretion of N-containing compounds increased, particularly when high levels of ammonium were added. Under these conditions, C, but not N, limited fungal growth. Although the overall production of mineral-associated organic C was not affected by ammonium concentrations, the observed shifts in the activities of the ex vivo and in vivo pathways affected the composition of organic matter adsorbed onto the mineral particles. Such changes will affect the properties of organic matter-mineral associations and, thus, ultimately, the stabilization of SOM.IMPORTANCE Nitrogen (N) availability plays a critical role in the cycling and storage of soil organic matter (SOM). However, large uncertainties remain in predicting the net effect of N addition on soil organic carbon (C) storage due to the complex interactions between organic matter, microbial activity, and mineral particles that determine the formation of stable SOM. Here, we attempted to disentangle the effects of ammonium on these interactions in controlled microcosm experiments including the ectomycorrhizal fungus P.involutus and dissolved organic matter extracted from forest soils. Increased ammonium levels affected the fungal processing of the organic material as well as the secretion of extracellular metabolites. Although ammonium additions did not increase the net production of mineral-adsorbed C, changes in the decomposition and secretion pathways altered the composition of the adsorbed organic matter. These changes may influence the properties of the organic matter-mineral associations and, thus, the stabilization of SOM.