Food contamination risks from mycotoxins pose a serious threat to public health. These toxic compounds are produced by filamentous fungi such as Aspergillus, Penicillium, Fusarium and Alternaria under specific temperature and humidity conditions. They can affect a wide range of agricultural products (nuts, cereals and their derivatives) during both the pre- and post-harvest stages. Among these, aflatoxin B1 is especially dangerous and has been classified as carcinogenic to humans. As part of the project PRIN 2022 PNRR “P20223P48S” entitled “Implementing advanced elemental and chemical analysis for quality, safety and traceability assessment of PGI and PDO agri-food products” funded by the European Union NextGenerationEU, we evaluated the feasibility of detecting aflatoxins in pistachio seeds using Fourier-Transform Infrared (FT-IR) spectroscopy. As a rapid and non-destructive technique, FT-IR holds significant potential for identifying contaminants in food, including mycotoxins. In the framework of the above project, FT-IR spectroscopy was initially applied to investigating the responses of pistachio samples from different locations in Sicily. The results demonstrate the ability of the FT-IR technique to discriminate between pistachios of different geographical origins, highlighting the connection between terroir and the chemical composition of the crops. Further studies are underway to evaluate the method’s potential to distinguish between contaminated and uncontaminated seeds by detecting the presence of aflatoxins B1, B2, G1, and G2. FT-IR analysis is being used to compare the chemical profiles of contaminated samples with those of mycotoxin-free ones.