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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-horizon 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 different hydrology and topographic position to parse patterns in chemistry and microbiota of the O-horizon in a north-temperate deciduous forest. There were frequent strong effects of tree species. Organic horizon properties under white ash frequently differed from the other trees: e.g., lower cation exchange capacity and exchangeable acidity, thinner Oi horizon, lower %C and C:N, and, from phospholipid fatty acids, more AM fungi and less gram positive 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 forest stands that we studied were established only 85—100 years ago. We also found patterns in the O-horizon related to underlying soil type, independent of tree species: e.g., Bh podzols, compared to typical podzols, had higher trace metals, thicker Oa horizon, and more AM fungi. Relationships between mineral soil type and the organic horizon could arise because landscape features that influence hydrology and therefore soil formation over centuries also influence biogeochemistry of the organic horizon over decades. It could also involve bioturbation by organisms across horizons. There is basic and applied value in better understanding of properties of the O-horizon based on vegetation and soil types.