Plant production systems that are more sustainable than conventional monoculture croplands are the vision of future agriculture. With numerous environmental benefits, agroforestry is among the most promising alternatives. Although soil fungi are key drivers of plant productivity and ecosystem processes, investigations of these microorganisms in temperate agroforestry systems are scarce, leaving our understanding of agricultural systems under agroforestry practice incomplete. Here, we assessed the composition and diversity of the soil fungal community as well as the frequency (relative abundance) of fungal groups in three paired temperate poplar-based alley cropping (agroforestry) and monoculture cropland systems by amplicon sequencing. Analysis of microbiomes using relative abundances of species or sequence variants obtained from amplicon sequencing ignores microbial population size, which results in several problems. For example, species stimulated by environmental parameters may appear unaffected or suppressed in amplicon counts. Therefore, we determined absolute abundances of selected fungal groups as well as total fungal population size by real-time polymerase chain reaction (PCR). Tree rows strongly affected the community composition and increased the population size and species richness of soil fungi. Furthermore, ectomycorrhiza were strongly promoted by the tree rows. We speculate that mycorrhiza improved the nutrient acquisition in unfertilized tree rows, thereby contributing to the total productivity of the system. Comparison of relative and absolute abundances revealed dramatic discrepancies, highlighting that amplicon sequencing alone cannot adequately assess population size and dynamics. The results of our study highlight the necessity of combining frequency data based on amplicon sequencing with absolute quantification.