Blood donation heavily depends on voluntary involvement, but the problem of motivating and retaining potential blood donors remains. Understanding the personality traits of donors can assist in this case, bridging communication gaps and increasing participation and retention. To this end, an eye-tracking experiment was designed to examine the viewing behavior of 75 participants as they viewed various blood donation-related advertisements. The purpose of these stimuli was to elicit various types of emotions (positive/negative) and message framings (altruistic/egoistic) to investigate cognitive reactions that arise from donating blood using eye-tracking parameters such as the fixation duration, fixation count, saccade duration, and saccade amplitude. The results indicated significant differences among the eye-tracking metrics, suggesting that visual engagement varies considerably in response to different types of advertisements. The fixation duration also revealed substantial differences in emotions, logo types, and emotional arousal, suggesting that the nature of stimuli can affect how viewers disperse their attention. The saccade amplitude and saccade duration were also affected by the message framings, thus indicating their relevance to eye movement behavior. Generalised linear models (GLMs) showed significant influences of personality trait effects on eye-tracking metrics, including a negative association between honesty–humility and fixation duration and a positive link between openness and both the saccade duration and fixation count. These results indicate that personality traits can significantly impact visual attention processes. The present study broadens the current research frontier by employing machine learning techniques on the collected eye-tracking data to identify personality traits that can influence donation decisions and experiences. Participants’ eye movements were analysed to categorize their dominant personality traits using hierarchical clustering, while machine learning algorithms, including Support Vector Machine (SVM), Random Forest, and k-Nearest Neighbours (KNN), were employed to predict personality traits. Among the models, SVM and KNN exhibited high accuracy (86.67%), while Random Forest scored considerably lower (66.67%). This investigation reveals that computational models can infer personality traits from eye movements, which shows great potential for psychological profiling and human–computer interaction. This study integrates psychology research and machine learning, paving the way for further studies on personality assessment by eye tracking.