Chronic elevated N deposition over the decades has altered the biogeochemistry of temperate forests in many areas and affected belowground biota. The Bear Brook Watershed in Maine (BBWM) is a paired watershed manipulation experiment where one watershed was treated bi-monthly with nitrogen (N) and sulfur (S) as ammonium sulfate [(NH₄)₂SO₄] for 27 years to study the impacts of elevated N and S deposition on forest function. Prior research at BBWM reported a negative fertilization effect on fungal:bacterial ratio, although no further research was conducted to evaluate these findings. We hypothesized that long-term N treatment could have caused a decline in mycorrhizal colonization in all tree species throughout the treated watershed. We investigated ectomycorrhizal (ECM) and arbuscular mycorrhizal (AMF) root colonization in American beech (Fagus grandifolia), sugar maple (Acre saccharum) and red spruce (Picea rubens) in both the treated and reference watersheds. Soil and root samples were collected by randomly selecting seven trees of each species within each watershed. From each tree, two samples were collected in a 10 x 10 cm frame from the upper 10 cm of O horizon. Soils were analyzed for various chemical variables. For quantification of mycorrhizal root colonization, the gridline intersect method was used with stained mycorrhizal roots to estimate proportion of colonization, and the total root length with the presence of a Hartig net for ECM and vesicles/arbuscles/hyphae inside the cortical cells for AMF. A boosted regression model was used to determine the relative influence of soil chemical variables on mycorrhizal colonization. Our results demonstrated that treatment with N and S has significantly reduced ECM colonization in red spruce with  83 ± 2.7% and 69 ± 1.8% colonization, and significantly reduced AMF in sugar maple with 65 ± 2.2% and 49 ± 2.1% colonization, in reference and treated watersheds, respectively, while there was no significant effects on mycorrhizal colonization in American beech. For both types of mycorrhizal colonization, exchangeable Al was found to be the most influential soil variable. Our hypothesis was supported by only two of the three tree species thereby indicating a host-specific mycorrhizal response to the treatment.  This study highlights the need to better understand the interaction between soil chemical variables, mycorrhiza, and their host in order to determine the effects of chronic N enrichment on forest ecosystem function.