A major environmental pollution problem is the release into the atmosphere of particulate matter, including nanoparticles (NPs), which causes serious hazards to human and ecosystem health, particularly in urban areas. However, knowledge about the uptake, translocation and accumulation of NPs in plant tissues is almost completely lacking. The uptake of silver nanoparticles (Ag-NPs) and their transport and accumulation in the leaves, stems and roots of three different tree species, downy oak (Quercus pubescens Willd.), Scots pine (Pinus sylvestris L.) and black poplar (Populus nigra L.), were assessed. In the experiment, Ag-NPs were supplied separately to the leaves (via spraying, the foliar treatment) and roots (via watering, the root treatment) of the three species. Uptake, transport and accumulation of Ag were investigated through spectroscopy. The concentration of Ag in the stem was higher in the foliar than in the root treatment, and in poplar more than in oak and pine. Foliar treatment with Ag-NPs reduced aboveground biomass and stem length in poplars, but not in oaks or pines. Species-specific signals of oxidative stress were observed; foliar treatment of oak caused the accumulation of H₂O₂ in leaves, and both foliar and root treatments of poplar led to increased O₂⁻ in leaves. Ag-NPs affected leaf and root bacteria and fungi; in the case of leaves, foliar treatment reduced bacterial populations in oak and poplar and fungi populations in pine, and in the case of roots, root treatment reduced bacteria and increased fungi in poplar. Species-specific mechanisms of interaction, transport, allocation and storage of NPs in trees were found. We demonstrated definitively that NPs enter into the tree stem through leaves faster than through roots in all of the investigated tree species.