A small library of six polarity-sensitive fluorescent dyes, nicknamed MediaChrom, was prepared. This class of dyes is characterized by a pyrimidoindolone core fitted out with a conjugated push-pull system and a carboxy linker for a conceivable coupling with biomolecules. The optimized eight-step synthetic strategy involves a highly chemo- and regioselective gold-catalyzed cycloisomerization reaction. The photophysical properties of MediaChrom dyes have been evaluated in-depth. In particular, the MediaChrom bearing a diethylamino as an electron-donating group and a trifluoromethyl as an electron-withdrawing group displays the most interesting and advantageous spectroscopic features (e.g., absorption and emission in the visible range and a good quantum yield). Promising results in terms of sensitivity have been obtained in vitro on this dye as a membrane/lipophilic probe and as a peptide fluorescent label.