Non-Profit Organizations (NPOs) find themselves in a very competitive environment, as everyday consumers are constantly exposed to numerous advertisements; thus, they must find ways to capture consumers’ attention. The objective of this study is to explore how the different elements (image, text, logo) of print advertisements of NPOs using different emotional appeals (positive and negative) of a familiar and unfamiliar brand influence donation behaviour and the attitude toward the ad (Aad). Using eye-tracking technology and a survey, we conducted two experiments, one with unfamiliar brands of NPOs and another with a familiar brand. The results showed the advertisement areas on which participants fixated and their relationship with participants’ attitude towards the advertisement and donation behaviour. For unfamiliar NPOs, the less time it took the participant to first fixate on the logo area, the more positive attitude toward the ad when the advertisement used a negative frame. Also, participants spent more time in the image area of negatively framed ads when they had a more positive attitude toward the ad. On the other hand, for a familiar brand, the time to first fixate on the logo area had a negative correlation with the donation behaviour, indicating that the less time it takes to first fixate on the logo area, the more participants chose to donate.