This study focuses on the characterization of bacterial and yeast species through their autofluorescence spectra. Lactic acid bacteria (“Lactobacillus” sp.), and yeast (“Saccharomyces” sp.) were cultured under controlled conditions and studied for variations in their autofluorescence, particularly in the area representative of tryptophan residues of proteins. The emission and excitation spectra clearly reveal that bacterial and yeast species can be differentiated by their intrinsic fluorescence with UV excitation. The possibility of differentiation between different strains of “Saccharomyces” yeast was also studied, with clear differences observed for selected strains. The study shows that fluorescence can be successfully used to differentiate between yeast and bacteria and between different yeast species, through the identification of spectroscopic fingerprints, without the need for fluorescent staining.