Tobacco constitutes a global emergency with totally preventable millions of deaths per year and smoking-related illnesses. Public service announcements (PSAs) are the main tool against smoking and by now their efficacy is still assessed through questionnaires and metrics, only months after their circulation. The present study focused on the young population, because at higher risk of developing tobacco addiction, investigating the reaction to the vision of Effective, Ineffective and Awarded antismoking PSAs through: electroencephalography (EEG), autonomic activity variation (Galvanic skin response—GSR- and Heart Rate—HR-) and Eye-Tracking (ET). The employed indices were: the EEG frontal alpha band asymmetry and the frontal theta; the Emotional Index (EI), deriving from the GSR and HR signals matching; the ET Visual Attention (VA) index, based on the ratio between the total time spent fixating an area of interest (AOI) and its area. Smokers expressed higher frontal alpha asymmetry values in comparison to non-smokers. Concerning frontal theta, Awarded PSAs reported the highest values in comparison to both Effective and Ineffective PSAs. EI results highlighted that lowest values were expressed by Heavy Smokers (HS), and Effective PSAs obtained the highest EI values. Finally, concerning the Effective PSAs, regression analysis highlighted a correlation between the number of cigarettes smoked by participants (independent variable) and frontal alpha asymmetry, frontal theta and EI values. ET results suggested that for the Ineffective PSAs the main focus were texts, while for the Effective and Awarded PSAs were the visual elements. Results support the use of methods aimed at assessing the physiological reaction for the evaluation of PSAs images, in particular when considering the smoking habits of target populations.