Pollution affects most of the urban and forest environments at different levels causing well-known effects on human and plant health. The influence that pollutants exert on plant-associated microbiota might direct plant health and, in some cases, also the removal of pollutants by plants. With the advent of nanotechnologies, an increasing amount of engineered nanoparticles are being introduced into the environment, and consequently, their impact on plant-associated microorganisms needs to be investigated. In this context, silver nanoparticles (Ag-NPs) were experimentally supplied at leaf and root level of poplar plants to assess Ag-NPs effects on plant microbiota. Leaf Ag-NP treatment increased bacteria and fungi evenness and determined a significant reduction in both microbial groups, while root Ag-NP treatment reduced the bacterial and fungal biodiversity. Bioinformatics functional analysis showed that Ag-NP treatment reduced the aerobic and stimulated facultative anaerobic and oxidative stress-tolerant bacteria. Our study offers new insights into the effects of Ag-NPs on both phyllosphere and rhizosphere poplar-associated microbiota and may represent a first attempt to understand the behavior of microbial communities of a tree species growing in a polluted environment.