The result of the enormous usage of pesticides in agriculture is the contamination of soil and water bodies surrounding the fields. Therefore, creating buffer zones to prevent water contamination is very useful. Chlorpyrifos (CPS) is the active substance of a number of insecticides widely used all over the world. In our study, we focused on the effect of CPS on plants forming riparian buffer zones: poplar (Populus nigra L., TPE18), hybrid aspen (P.tremula L. × P. tremuloides Michx.), and alder (Alnus glutinosa L.). Foliage spray and root irrigation experiments were conducted under laboratory conditions on in vitro cultivated plants. Spray applications of pure CPS were compared with its commercially available form—Oleoekol®. Although CPS is considered a nonsystemic insecticide, our results indicate that CPS is transferred not only upwards from roots to shoots but also downwards from leaves to roots. The amount of CPS in the roots was higher (4.9 times and 5.7 times, respectively) in aspen or poplar sprayed with Oleoekol than in those sprayed with pure CPS. Although the treated plants were not affected in growth parameters, they showed increased activity of antioxidant enzymes (approximately two times in the case of superoxide dismutase and ascorbate peroxidase) and augmented levels of phenolic substances (control plants −114.67 mg GAE/g dry tissue, plants treated with CPS—194.27 mg GAE/g dry tissue). In summary, chlorpyrifos, especially as a foliar spray pesticide, can create persistent residues and affects not only target plants but also plants surrounding the field.