A long-term horticultural experiment was conducted at two geographically distinct sites in southern Missouri in 2011–15 to study the response of American elderberry [Sambucus nigra (L.) subsp. canadensis (L.) Bolli] to various soil nitrogen (N) fertilizer levels. Three commercially available elderberry cultivars (‘Adams II’, ‘Bob Gordon’, and ‘Wyldewood’) were used. The three cultivars were each assigned to 16 of 48 four-plant plots in a completely randomized manner at each site. Four replications of four N fertilizer treatments (0, 56, 112, 169 kg⋅ha−1 N) were randomly assigned to each cultivar’s plots and applied for 4 years (2012–15). Fruit yields, plant growth, phenology, and pest incidence were determined each year. Fruit quality was assessed by analyzing basic juice characteristics as well as organic acids, carbohydrates, anthocyanins, and polyphenols from 2012–14 samples. Leaf tissue analysis determined the plants’ mineral contents in 2012–14. Most factors evaluated were significantly affected by site, year, and cultivar, whereas the effects of N fertilizer treatment were less definitive. Fruit yields and plant growth increased with increasing N levels. For example, plants fertilized with 0, 56, 112, and 169 kg⋅ha−1 N produced 123, 137, 155, and 161 fruiting cymes per plot (5.8 m2), respectively. The eriophyid mite incidence was higher on fertilized plants, but other pests were not influenced by the N treatment. Basic fruit juice characteristics (soluble solids, pH, titratable acidity, polyphenols) were not influenced by the N treatment, whereas total anthocyanins were statistically higher in unfertilized plants. Levels of organic acids and carbohydrates in juice varied statistically among N treatments, but patterns were difficult to discern. Leaf N concentrations were correlated with N fertilizer levels—2.75% N with the highest fertilizer level compared with 2.55% N in unfertilized plants. Leaf levels of most other macronutrients varied, but consistent patterns did not emerge, and none of the micronutrients was different among N treatments. Although elderberry plants responded positively to increased N fertilizer levels in terms of plant growth and fruit yield, genetics (cultivar) and environment (site, year) were more influential on most other experimental factors evaluated.