The inversion of a picture of a face hampers the accuracy and speed at which observers can perceptually process it. Event-related potentials and pupillary responses, successfully used as biomarkers of face inversion in the past, suggest that the perception of visual features, that are organized in an unfamiliar manner, recruits demanding additional processes. However, it remains unclear whether such inversion effects generalize beyond face stimuli and whether indeed more mental effort is needed to process inverted images. Here we aimed to study the effects of natural scene inversion on visual evoked potentials and pupil dilations. We simultaneously measured responses of 47 human participants to presentations of images showing upright or inverted natural scenes. For inverted scenes, we observed relatively stronger occipito-temporo-parietal N1 peak amplitudes and larger pupil dilations (on top of an initial orienting response) than for upright scenes. This study revealed neural and physiological markers of natural scene inversion that are in line with inversion effects of other stimulus types and demonstrates the robustness and generalizability of the phenomenon that unfamiliar configurations of visual content require increased processing effort.