Abstract: The development of new NIR fluorescent probes is a very important issue due to the wide range of applications. Benzo[a]phenoxazines are molecules with fluorescence at around 600 nm and have been reported also as antifungal agents. Herein, the synthesis of four new Nile Blue derivatives with hydrogen, propyl and/or aminopropyl groups as substituents of the amines of 5- and 9-positions is described. Photophysical properties are evaluated in acidified ethanol and aqueous solution at physiological pH. Antifungal activity is also evaluated through the obtention of MIC values.