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(1) Background: Why and when images of watching eyes encourage prosocial behavior is still subject to discussion, and two recent meta-analyses show no effect of watching eyes on generosity. This study aims to discern the effect of watching eyes of different valence on two separate aspects of prosocial behavior, and additionally investigates whether individuals' social value orientation moderates the effect of eyes. (2) Methods: Individuals take on the role of either a first or second mover in an incentivized, anonymous sequential prisoner's dilemma (n = 247), a two-person game which separates the need to form expectations about the other player (first mover cooperation, trust) from the motive of greed (second mover cooperation, reciprocity). During decision-making, a picture of either kind eyes, unkind eyes, or a control picture is presented above each decision matrix. (3) Results: The results indicate that unkind eyes, and not kind eyes, significantly boost first mover cooperation. In contrast, neither type of eye cues increase second mover cooperation. Social value orientation does not moderate these effects. (4) Conclusions: Thus, the data suggest that the valence of eye cues matters, and we propose that unkind eyes urge first movers to live up to the interaction partner's expectations.