The fluorescence and phosphorescence behavior of the fluorone dyes disodium fluorescein (uranine), 4,5-dibromofluorescein, eosin Y, erythrosine B, and rose bengal in biofilms of chitosan, gelatin, and starch was studied in the temperature range from 293 K to 12 K. Luminescence quantum yields, fluorescence quantum yields, and triplet quantum yields were determined. The fluorescence quantum yield generally increased with lowering the temperature at a high level of absolute quantum yield, while the phosphorescence quantum yield increased with decreasing temperature at a low level of absolute quantum yield. The strongest phosphorescence was found for erythrosine B in starch where the phosphorescence quantum yield increased from 6% at 293 K to 25% at 12 K. The temperature dependent luminescence behavior is discussed considering radiative and non-radiative transitions in a spectroscopic-state potential energy surface scheme.