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Interactions between above- and below-ground monoculture forest plantation components are critical to tree growth and development. Within the Central Hardwoods Region (CHR), synergistic relationships between tree species and soil microbial community structure and function have received limited research attention. Soil microbes are integral to forest ecosystems as their activities intrinsically promote soil organic matter decomposition, nutrient cycling, and ecosystem functioning. Here, we examined soils from two perfectly aligned stands of black walnut (BW, Juglans nigra L.) and Northern red oak (RO, Quercus rubra L.) trees. Measurements of selected soil chemical properties, microbial community structure using ester-linked fatty acid methyl ester (EL-FAME), and soil enzyme activities (EAs) were used. Analysis of modifications within microbial communities showed a significant positive response to BW based upon soil EAs and microbial indicators, compared to RO. Seasonal comparisons predictably revealed higher microbial activities during summer. Fungi dominated the soil microbial community structure with a fungal/bacterial ratio of 2:1. Gram-positive rather than Gram-negative bacteria or actinomycetes dominated the bacterial community. The activity of the soil enzymes ß-glucosidase and arylsulfatase increased, but ß-glucosaminidase and acid phosphatase decreased. Additionally, acid phosphatase and arbuscular mycorrhizal fungi revealed strong correlations. The differences observed in biological properties, specifically microbial communities and EAs, highlight the varied responses to BW and RO soil biology and subsequent soil ecosystem functions. These results indicate that variations in microbial abundance and soil functions occur throughout the course of an entire year.