Plant pathogens are an important agricultural problem, and early and rapid pathogen identification is critical for crop preservation. This work focuses on using fluorescence spectroscopy to characterize and compare healthy and fungal pathogen-infected wheat grains. The excitation–emission matrices of whole wheat grains were measured using a fluorescence spectrometer. The samples included healthy control samples and grains manually infected with Fusarium graminearum and Alternaria alternata fungi. The five distinct zones were identified by analyzing the location of the fluorescence peaks at each measurement. The zone centered at λem = 328/λex= 278 nm showed an increase in intensity for grains infected with both pathogens during all periods of the experiment. Another zone with the center λem = 480/λex = 400 nm is most interesting from the point of view of early diagnosis of pathogen development. A statistically significant increase of fluorescence for samples with F. graminearum is observed on day 1 after infection; for A. alternata, on day 2, and the fluorescence of both decreases to the control level on day 7. Moreover, shifts in the emission peaks from 444 nm to 452 nm were recorded as early as 2–3 h after infection. These results highlight fluorescence spectroscopy as a promising technique for the early diagnosis of fungal diseases in cereal crops.