The objective of the present study was to assess the relative contributions of microbial (mineralization of non-microbial organic phosphorus and microbial phosphorus) and physical–chemical (diffusion of phosphate ions at the solid-to-solution interface) processes involved in replenishing the soil solution with phosphate ions. This assessment was carried out across soil depths and subsystems in a forest ecosystem. Major differences in the contribution of microbial processes (CMP%) were found between soil layers and subsystems, corresponding with differences in soil organic matter and Al and Fe oxide contents. CMP% significantly decreased with increasing soil depth (from 87–100% in the forest floor to 2–16% in the 90–120 cm mineral soil layer). Tree fine roots were mostly concentrated in the top soil layers where microbial processes dominated. Consequently, estimates of CMP% in the entire soil profile were also affected by the vertical distribution of tree fine roots. We assumed that including fine root densities leads to a more correct estimation of the role of microbial processes (CMP% = 31–52% in the entire soil profile, up to 75% in one particular site). To our knowledge, this is the first assessment of the relative contributions of the different processes involved in replenishing the soil solution with phosphate ions based on soil properties and fine root distribution.