Laboratory studies have shown that images of eyes can cause people to behave more cooperatively in some economic games, and in a previous experiment, we found that eye images increased the level of contributions to an honesty box. However, the generality and robustness of the eyes effect is not known. Here, we extended our research on the effects of eye images on cooperative behavior to a novel context—littering behavior in a university cafeteria—and attempted to elucidate the mechanism by which they work, by displaying them both in conjunction with, and not associated with, verbal messages to clear one's litter. We found a halving of the odds of littering in the presence of posters featuring eyes, as compared to posters featuring flowers. This effect was independent of whether the poster exhorted litter clearing or contained an unrelated message, suggesting that the effect of eye images cannot be explained by their drawing attention to verbal instructions. There was some support for the hypothesis that eye images had a larger effect when there were few people in the café than when the café was busy. Our results confirm that the effects of subtle cues of observation on cooperative behavior can be large in certain real-world contexts.