Background <p>Direct total-DNA (tDNA) extraction from soil recovers both cell-associated and extracellular (relic) DNA, which can distort amplicon-based views of microbial communities; how strongly this biases bacteria versus fungi, and whether it varies with soil depth, remains poorly resolved for cultivated soils. Using a paired split-sample design, we compared bacterial 16S rRNA gene and fungal ITS profiles from directly extracted tDNA and an operationally defined intracellular-DNA-enriched (iDNA) fraction in bulk soil sampled at three depths from a continuously cultivated Lonicera japonica field.</p> Results <p>The two DNA pools gave related but clearly non-interchangeable pictures of the community, and the two kingdoms responded asymmetrically: bacteria were consistently the more sensitive, with higher richness recovered from tDNA and a marked separation between DNA pools throughout the profile, whereas the fungal contrast was smaller overall and, for alpha diversity and within-pair divergence, concentrated in the surface soil. DNA-pool choice acted mainly by selectively redistributing specific lineages rather than replacing the dominant community, while the measured soil physicochemical variables explained only a small share of community variation within either pool.</p> Conclusions <p>Direct total DNA and an iDNA-enriched fraction offer kingdom- and depth-dependent windows on the same soil, so the choice of DNA pool is a consequential design decision—especially for bacteria and for surface horizons—that should be matched to the ecological question and independently validated when a cell-associated fraction is the goal. More broadly, a single total-DNA snapshot cannot be assumed to represent the cell-associated community equally across taxa or depths; treating the DNA-extraction strategy as an ecological variable rather than a neutral technical step would strengthen the comparability and interpretation of bulk-soil bacterial and fungal community surveys.</p>