The study of nitrogen organic matter is of great importance for both economic and environmental reasons. Thus, seven soils developed under different pedogenetic and bioclimatic conditions in Tunisia were selected to investigate the distribution of organic nitrogen (ON). Our results showed an enrichment in ON of superficial horizons to the detriment of the deep horizons. Nevertheless, this decrease in ON contents with depth occurred following different patterns according to soil type. Moreover, the investigation of ON distribution in clay- and silt-sized particles (below 50 µm) indicated a considerable contribution of the fine fraction (65–98% of the soil total ON) to the ON immobilization in soils, especially in the deep horizons, while the coarse fraction (>50 µm) seems to play a less important role. The isolation of humic substances (Humic Acids, Fulvic Acids and Humin (H)) from the fine fraction and the analysis of the ON in each of these organic fractions permitted to conclude the majority of soil ON is incorporated within the H, thus suggesting a low bioavailability of nitrogen in the studied soils.