A research project, funded by the Italian Ministry of Research and the European Union, is in progress aimed to develop processes to enhance, by irradiation, the safety and the wholesomeness of fresh products relevant for Italian food industry. Irradiation was performed on truffles, since the bacterial contamination impairs their trade in foreign countries. The microbial population and the shelf life under refrigeration were studied either on samples untreated or on samples submitted to g-rays in a 1–2.5 kGy dose range. The effect of the treatment was monitored by UV and NMR techniques. Total microbial population and the shelf life prolongation were investigated. The synergistic effect of the dose, the packaging under vacuum and the storage/irradiation temperature resulted in a direct effect on the microbial load, spoilage and shelf life. After the irradiation, small variations in the intensity of some NMR resonances due to aromatic compounds and other unassigned compounds were observed. As confirmed by UV spectrophotometric data, these phenomena seemed to originate from a small degradation of polyphenols; the induced growth of soluble phenols suggested that the 1.5 kGy dose can be considered as the radiation dose threshold beyond which clear chemical modifications on truffles appear.