The fluorescence lifetime of ethidium bromide (EB), a widely used fluorescent dye, has been monitored in water solution versus trehalose concentration in order to learn how the presence of the disaccharide modifies the hydration of EB, which is used in this study as a model probe. The interest in trehalose, a naturally occurring bioprotector (osmolyte), stems from its ability to stabilize biomolecules under stress situations. The observed effects of trehalose on EB fluorescence properties have been compared with those induced by sucrose and glucose. Lifetime measurements have been performed by frequency domain fluorometry (2–40 MHz), and from the analysis of their changes versus sugar concentration, the EB–sugar interaction parameters have been obtained. The effect of trehalose on the EB decay in glasses has also been studied both by exploring the heterogeneity of lifetime decay and by single-molecule imaging. Trehalose appears to be more efficient in changing the EB fluorescence parameters, such as the emission lifetime, and it leads to a degree of heterogeneity higher than that induced by the other sugars. When EB is embedded in trehalose glasses, the heterogeneity of the emission and of the bleaching time is further enhanced.