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As the interface between plants and soil, the organic horizon is the foundation of forest ecosystems. Two potential predictors of O-layer properties, vegetation and mineral soil type, are difficult to separate because they typically covary. We conducted a factorial study involving four canopy tree species and two soil types with distinctly different hydrology and topographic position to parse patterns in chemistry and microbiota of the O-layer in a north-temperate deciduous forest. There were frequent strong effects of tree species. White ash frequently differed from the other trees: e.g., lower cation exchange capacity and exchangeable acidity, thinner Oi layer, lower %C and C:N, and, from phospholipid fatty acids, more AM fungi and less gram+ bacteria. These patterns, presumably due to species-specific attributes of leaf litter quality, root exudates, and microbial associations, must arise over decades, given that the stands in the study age between 85 and 100 years. We also found patterns in the O-layer related to underlying soil type, independent of tree species: e.g., Bh podzols, compared to Typical podzols, had higher trace metals, thicker Oa layer, and more AM fungi. Relations between mineral soil type and the organic layer, which were larger than expected, could arise because landscape features that influence hydrology and therefore soil formation over millennia also influence biogeochemistry of the organic layer over decades. It could also involve bioturbation by organisms across horizons. There is basic and applied value in models that can predict properties of the O-layer based on vegetation and soil types.