Tsuga canadensis (Eastern Hemlock) is a key forest foundation species that is currently declining across the eastern US due to attack by exotic insect species, primarily Adelges tsugae (Hemlock Woolly Adelgid). In the northeastern US, declining Eastern Hemlock stands are typically replaced by fast-growing deciduous Betula lenta (Black Birch) trees, altering ecosystem processes and ecological communities. In this study, we used an approach that substituted space for time to explore how the forest floor's soil organic horizon, macrofungal communities, and bacterial abundance might be altered following Eastern Hemlock replacement by Black Birch. We compared intact, mature Eastern Hemlock forest plots at 2 sites in western Massachusetts to adjacent areas of vigorous regeneration of young Black Birch triggered by logging activity at the sites ∼25–30 years ago. Forest-floor soil organic horizons were significantly thicker under Eastern Hemlock forests and exhibited a higher carbon:nitrogen (C:N) ratio, suggesting slower decomposition and greater accumulation of organic material under Eastern Hemlock compared to deciduous Black Birch canopies. Macrofungal communities emerging from the forest floor did not differ strongly in morphospecies richness between Eastern Hemlock and Black Birch plots. However, a greater number of rare fungi taxa, defined in this study as those represented by a single observation, were detected in Eastern Hemlock plots, and the composition of Black Birch plots was more homogenized and less variable. The abundance of bacterial colony forming units (CFUs) in the soil organic horizon appeared to follow a seasonal pattern of variation between mature Eastern Hemlock versus young Black Birch plots; CFUs were most abundant in Black Birch soils in the fall, potentially tracking input of new deciduous leaf litter, whereas Eastern Hemlock plots had higher CFU counts in the summer. The results of this study suggest that forest-floor characteristics will be substantially altered by the impending loss of Eastern Hemlock, associated macrofungal communities may become simplified and homogenized, and the timing of peak bacterial abundance in the forest floor might be shifted to the fall.