Cholinergic pesticides, such as the neonicotinoid imidacloprid, are the most important insecticides used for plant protection worldwide. In recent decades, concerns have been raised about side effects on non-target insect species, including altered foraging behavior and navigation. Although pollinators rely on visual cues to forage and navigate their environment, the effects of neonicotinoids on visual processing have been largely overlooked. To test the effect of acute treatment with imidacloprid at known concentrations in the brain, we developed a modified electrophysiological setup that allows recordings of visually evoked responses while perfusing the brain in vivo. We obtained long-lasting recordings from direction selective wide-field, motion sensitive neurons of the hoverfly pollinator, Eristalis tenax. Neurons were treated with imidacloprid (3.9 μM, 0.39 μM or a sham control treatment using the solvent (dimethylsulfoxide) only. Exposure to a high, yet sub-lethal concentration of imidacloprid significantly alters their physiological response to motion stimuli. We observed a general effect of imidacloprid (3.9 μM) increasing spontaneous activity, reducing contrast sensitivity and giving weaker directional tuning to wide-field moving stimuli, with likely implications for errors in flight control, hovering and routing. Our electrophysiological approach reveals the robustness of the fly visual pathway against cholinergic perturbance (i.e., at 0.39 μM) but also potential threatening effects of cholinergic pesticides (i.e., evident at 3.9 μM) for the visual motion detecting system of an important pollinator.