Fluorescence methods are widely used for investigation of biological materials, and in recent years have also been used to monitor food quality and safety. In this research, fluorescence imaging techniques for detecting fecal contamination on cantaloupes and strawberries were evaluated. Fluorescence images at emission peaks were examined for fecal classification. These images were subjected to further analysis utilizing band ratios and principal component analysis. Two-band ratio images and principal component images, compared to the single-band images, enhanced the contrast between the feces-contaminated spots and untreated sample surfaces. The images exhibited useful results for contamination detection, however, false positives resulting from natural color variation on strawberry surfaces present a problem throughout the methods. This study confirmed the capability of hyperspectral fluorescence imaging in detecting fecal matter on cantaloupes and strawberries and the potential for this method to be used for developing on-line applications.