Cooperation is essential in social life, involving collaborative efforts for mutual benefit. Individual differences in the cooperativeness trait are pivotal in these interactions. Previous research suggested that Duchenne smiles signal cooperative intent, and human-like eyes are theorized to enhance cooperation evolutionarily. However, the relationships between cooperative behavior, cooperativeness and possible behavioral cues signaling cooperative behavior are yet to be studied. In the current study, we explored the relationships among cooperativeness, Duchenne smiling with gaze, IBS during conversation, and their impacts on cooperative behavior. We hypothesized Duchenne smiling with gaze would mediate cooperativeness’ impact on cooperative behavior. Additionally, using functional near-infrared spectroscopy (fNIRS), we evaluated inter-brain synchrony (IBS) in the left prefrontal region, expecting it to predict cooperative behavior. The results demonstrated that cooperativeness significantly predicted Duchenne smiling with gaze and cooperative behavior, however, Duchenne smiling with gaze did not mediate the relationship between them. We further conducted a path analysis to examine the role of IBS during conversation in successive cooperative behavior. The path analysis result showed that cooperativeness directly affected cooperative behavior. Cooperativeness significantly predicted Duchenne smiling with gaze, however, neither Duchenne smiling with gaze nor IBS during conversation predicted successive cooperative behavior. These results suggest dispositional factors like cooperativeness may play a more decisive role than momentary expressional cues or neural synchrony in naturalistic unstructured communication in shaping cooperative behavioral outcomes after the communication. The study highlights how personality traits like cooperativeness shape nonverbal communication and social interactions.