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Background and aims: Phosphorus (P) is an essential element for crop growth. However, while links of P turnover in soils to carbon (C) and nitrogen (N) availability have been described, it remains to be clarified how combinations of fertilizer C and N additions affect stocks and cycling of distinct P fractions at different soil depths. The objectives of our study were (1) to assess how soil total P stocks are affected by organic amendments and N fertilization, (2) to evaluate how different soil P fractions respond to N fertilization, and (3) to verify whether N fertilization increases soil biological P cycling. Methods: We collected soil samples from a long‐term field experiment established in 1984 in Rauischholzhausen, Germany. The soil is a Haplic Luvisol and received either no organic fertilizer (NOF), farmyard manure (FYM) or a combination of organic and mineral N fertilizer (OMF). Each treatment additionally received three levels of mineral N: 0 kg ha−1 y−1 (N0), 100 kg ha−1 y−1 (N100), and 200 kg ha−1 y−1 (N200). The organic fertilizers were applied by a manure spreader and the N fertilizer (calcium ammonium nitrate) was applied in spring as top dressing by a plot fertilizer machine. We estimated stocks of P in fractions isolated by sequential P fractionation, and assessed the oxygen isotopic composition of 1 M HCl‐extractable phosphate (δ18OP). Results: We found that increased organic matter (OM) addition and mineral N inputs caused significant decreases in the stocks of resin‐ and NaHCO3‐extractable P in the topsoil (0–30 cm). Mineral N fertilization alone resulted in significant increases in stocks of resin‐, NaHCO3‐, and NaOH‐extractable P in the upper subsoil (30–50 cm). These changes occurred for both inorganic and organic P. The subsoil δ18OP values were closer to expected equilibrium values in soil fertilized with mineral N, indicative of more intensive biological P cycling than in the treatments without mineral N inputs. Conclusions: These findings suggest that long‐term OM and mineral N fertilization promotes topsoil P losses from labile fractions by crop uptake with an enrichment of these P forms in the subsoil, and an overall increase in biological P cycling in both top‐ and subsoil horizons upon N fertilization.