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The plant microbiome is an important factor for plant health and productivity. While the impact of nitrogen (N) availability for plant growth and development is well established, its influence on the microbial phyllosphere community structure is unknown. We hypothesize that nitrogen impacts the growth and abundance of several microorganisms on the leaf surface. The bacterial and fungal communities of baby leaf spinach (Spinacia oleracea), and rocket (Diplotaxis tenuifolia) were investigated in a field trial for two years in a commercial setting. Nitrogen fertilizer was tested in four doses (basic nitrogen, basic + suboptimal, basic + commercial, basic + excess) with six replicates in each. Culture-independent (Illumina sequencing) and culture-dependent (viable count and identification of bacterial isolates) community studies were combined with monitoring of plant physiology and site weather conditions. This study found that alpha diversity of bacterial communities decreased in response to increasing nitrogen fertilizer dose, whereas viable counts showed no differences. Correspondingly, fungal communities of the spinach phyllosphere showed a decreasing pattern, whereas the decreasing diversity of fungal communities of rocket was not significant. Plant species and effects of annual variations on microbiome structure were observed for bacterial and fungal communities on both spinach and rocket. This study provides novel insights on the impact of nitrogen fertilizer regime on a nutrient scarce habitat, the phyllosphere., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)