<p>Ultrastructural observation of the rhizosphere requires preservation of spatial continuity across heterogeneous biological and mineral components. Although correlative imaging approaches integrate electron microscopy with chemical and isotopic analyses, maintaining large-area structural integrity in intact plant–microbe–soil composites remains technically demanding. Here, we show that biological cross-sectional polishing scanning electron microscopy enables centimeter-scale cross-sectional observation of resin-embedded soil interfaces while retaining subcellular ultrastructural detail. The workflow combines conventional fixation used for plant electron microscopy with low-viscosity resin embedding, diamond band-saw sectioning, and mechanical polishing to generate flat cross sections suitable for backscattered electron imaging using standard scanning electron microscopes. Application to laboratory-grown and field-collected samples revealed microbial aggregates, biofilm-like matrices, fungal networks, and diverse soil-associated organisms observed within preserved spatial relationships. Although this method does not provide chemical or lineage-specific identification, it establishes a reproducible structural framework that complements multiscale imaging and supports targeted downstream analyses in complex environmental systems.</p><p></p>