Aim: To assess the influence of elastomeric ligatures, subjected to a previous in vitro pigmentation process using different substances, on smile aesthetics during orthodontic treatment, from the perception of students and professionals. Methods: Eight elastomeric ligatures of five commercial brands (3M/Unitek, American Orthodontics, Morelli, Ortho Technology, and Orthometric) (n=8) were immersed in coffee, Coca-Cola, and red wine for one minute per day, for 28 days; and another group of ligatures was immersed in artificial saliva. All samples were photographed and subsequently analyzed using the Adobe Photoshop software, by the RGB method. Afterwards, the pigmented ligatures were inserted in a patient wearing orthodontic brackets, and zoomed photographs of the smile were taken and presented to 40 evaluators, who filled in a satisfaction scale sheet to express their opinion on the smile aesthetics of each photograph. The color data were subjected to analysis of variance (ANOVA) and Tukey tests. Results: The substance with the highest pigmentation potential was coffee (p< 0.05) followed by red wine (p< 0.05). Comparison among the brands used in this study showed that American Orthodontics and Orthometric had the lowest degree of pigmentation when immersed in coffee and red wine (p< 0.05), respectively. However, the brand that showed the highest level of satisfaction among the evaluators was Ortho Technology. Conclusions: The presence of pigmented elastomeric ligatures affected smile aesthetics, when compared with the control group.