Numerous studies have demonstrated a link between neuroticism and negative biases. Although some studies suggest that people with high neuroticism give more weight to negative information, others suggest that they respond more strongly to both positive and negative information. We investigated whether neuroticism is related to the evaluation of conditioned stimuli (CSs) in evaluative conditioning procedures that involve ambiguous learning conditions. We created ambiguous situations where CSs were paired with unconditioned stimuli (USs) consisting of both positive and negative pictures (Experiment 1) or paired alternatingly with positive and negative USs (Experiment 2). In addition to CSs consistently paired with positive and negative USs, we introduced neutral USs as a control condition. Our findings revealed that neurotic individuals negatively evaluated the CSs from ambiguous conditions relative to neutral conditions. In addition, participants with high neuroticism scores generally rated CSs more negatively. Theoretical and clinical implications of these results are discussed.