Benzophenoxazine fluorophores, including derivatives of Nile Blue, one of the best known in this family of compounds, have been used as histological stains due to their optical properties in the NIR region and stability. Given their potential, the synthesis of two new Nile Blue derivatives with different substituents on the amines of the positions 5 and 9 was performed, with the introduction of a sulfonamide group in one of the compounds. Photophysical properties of the compounds were evaluated in acidified ethanol and aqueous solution at physiological pH. The fluorescent staining capacity was evaluated by staining Saccharomyces cerevisiae with the compounds, followed by fluorescence microscopy.