Phosphorus (P) availability shows large differences among different soil types, affecting P nutrition of forest trees. Chemical binding of P to soil moieties affects partitioning of P between soil particles and solution, influencing soluble P concentrations upon which plants, their associated mycorrhizal symbionts, and microbes feed. The goal of this study was to characterize root P uptake by mycorrhizal and non-mycorrhizal root tips in competition with microbes in situ in the organic and mineral layer of a P-rich and a P-poor forest. We used intact soil cores (0.2 m depth) from beech (Fagus sylvatica) forests to tracing the fate of 33 P in soil, plant and microbial fractions. We used the dilution of 33 P in the rhizosphere of each soil layer to estimate the enrichment with new P in mycorrhizal and non-mycorrhizal root tips and root P uptake. In soil cores from P-rich conditions, 25 and 75% of root P uptake occurred in the organic and mineral layer, respectively, whereas in the P-poor forest, 60% occurred in the organic and 40% in the mineral layer. Mycorrhizal P efficiency, determined as enrichment of new P in mycorrhizal root tips, differed between soil layers. Root P uptake was correlated with mycorrhizal P efficiency and root tip abundance but not with root tip abundance as a single factor. This finding underpins the importance of the regulation of mycorrhizal P acquisition for root P supply. The composition of mycorrhizal assemblages differed between forests but not between soil layers. Therefore, differences in P efficiencies resulted from physiological adjustments of the symbionts. Non-mycorrhizal root tips were rare and exhibited lower enrichment with new P than mycorrhizal root tips. Their contribution to root P supply was negligible. Microbes were strong competitors for P in P-poor but not in P-rich soil. Understory roots were present in the P-rich soil but did not compete for P. Our results uncover regulation of mycorrhizal P efficiencies and highlight the complexity of biotic and abiotic factors that govern P supply to trees in forest ecosystems.