While there are estimates of the abiotic processes contribution to soil phosphorus (P) availability, less is known about the contribution of biological processes. Two main enzymatic processes involved in soil P cycling are known to alter the oxygen isotopic composition of phosphate (δ18O-P), each in a different way, through the cleavage of the P–O bond: the intracellular P turnover and the organic P hydrolysis. The former induces isotopic equilibration between phosphate and water and is considered the major process affecting soil available P via microbial P release. The latter induces depleted δ18O-P in the phosphate released from the mineralization of organic P. We studied P dynamics in organic horizons of two contrasting soils (low- and high-P availability) from temperate beech forests. We labeled the soil with 18O-enriched water and followed changes in the δ18O-P of different soil P pools in the presence or absence of added leaf litter during 3 months of incubation. δ18O-P values of almost all P pools progressively increased indicating oxygen incorporation from the enriched soil water into phosphate via the above-mentioned enzymatic processes. δ18O-P of available P increased more in the P-rich soil than in the P-poor soil and approached the isotopic equilibrium between phosphate and water, revealing the impact of microbial P release into the available P pool. However, in the P-poor soil, the available P brought the isotopic signature induced by phosphatase enzymes, indicating that it was mostly originated from the hydrolysis of organic P. Therefore, under P-limiting conditions, the isotopic effect of organic P hydrolysis can outweigh the isotopic equilibrium effect. Finally, two independent isotopic approaches with 33P and δ18O-P provided very similar estimates of P exchanged between the available P and other inorganic soil pools. This suggests that δ18O-P can be successfully used to trace P fluxes, provided that the underlying processes do not break the P–O bonds of the phosphate molecule.