The number of Non‐Profit Organisations (NPOs) has increased in the past years, and they rely on advertising to communicate their causes. Although the effectiveness of NPO advertising has been studied previously, there is still no consensus about which appeal (emotionally positive or negative) increases its effectiveness. On the other hand, the most prevalent research approaches to NPO advertising are traditional research methodologies based on declarative techniques. So, the purpose of this study is to identify which appeal (positive or negative) in NPO advertising is more effective at the three levels of effectiveness (perceptual or communicational effectiveness, psychological or attitudinal effectiveness and behavioural effectiveness), providing a new approach based on the analysis of consumers' unconscious responses to advertising. We conducted an experiment with 113 participants whose neurophysiological responses were evaluated through electroencephalogram (EEG) and eye tracking (ET) while they watched NPO advertisements with a randomly assigned emotional appeal (positive or negative). In addition, a survey revealed behavioural responses. The results showed that considering the psychological or attitudinal effectiveness, positively framed ads are more effective, as there is a positive effect on attitudes towards the ad and a higher positive emotional valence. On the other hand, in perceptual effectiveness, where attention is considered an important variable, the negatively framed ads showed more significant time in the area of interest (AOI) of the image area of the ad, and longer time in the AOI text was observed for positively framed ads. Furthermore, regarding behavioural effectiveness, negatively framed ads seem more effective in eliciting actual donations. The results suggest that a positive appeal is more effective in generating a more positive attitude and a positive emotional valence towards the advertisement, which could benefit the NPO in the long term. But a negative appeal is more effective if the goal is to achieve immediate donations.