Abstract Background Mushrooms have always been considered an important source of food and biologically active compounds with several medicinal properties. In recent years, different methods were used to study the quality and chemical composition of mushrooms. Among these, Fourier transform infrared (FT-IR) and FT-Raman spectroscopy techniques have been successfully applied to identify different mushroom species. However, the structural biomolecule components existing in the mycelium or in the fruiting bodies may produce strong fluorescence emission that overlaps the Raman radiation, thus avoiding their analyses by Raman. SERS spectroscopy is a powerful technique which uses metal nanoparticles (NPs) to enhance the Raman signal of molecules adsorbed on the NPs surface. In addition, SERS is able to quench the macromolecule florescence. In this work, we have employed silver nanoparticles in order to get mushroom fingerprints based on SERS as quick procedure to analyze and identify different chemical compounds from the fruiting bodies of six edible and/or medicinal mushrooms: Lentinula edodes, Ganoderma lucidum, Pleurotus cornucopiae, Pleurotus ostreatus, Tuber aestivum and Tuber magnatum. Results SERS analyses performed directly on fruiting body fragments produced characteristic spectra for each species. One group of mushrooms (L. edodes, G. lucidum, T. aestivum and T. magnatum) was dominated by the bands of nucleic acids; and the other one (P. cornucopiae and P. ostreatus), by the bands of pigments such as melanins; carotenoids; azafilones; polyketides; and flavonoids located in the cell wall. Additionally, bands corresponding to cell wall polysaccharides, particularly chitosan and 1,3-β D-glucan, were identified in the extracts of P. cornucopiae, P. ostreatus and L. edodes. No signal of cell wall polysaccharides was found in G. lucidum extract. Raman mapping of the analyzed samples was useful in tracking the spatial distribution of the marker bands. Moreover, the principal component analysis (PCA) carried out on the acquired SERS spectra, allows to discriminate the analyzed mushroom species. Conclusions The SERS technique has the ability to generate a strong Raman signal from mushroom fruiting bodies using Ag-NPs deposited directly on intact, untreated mushroom tissues. Using this methodology, commonly applied laboratory time-consuming methods can be avoided or bypassed as well as analysis time can be reduced. Graphical Abstract