The investigation about archaeological samples, which are of inestimable value, requires minimally destructive or nondestructive sampling techniques. One of the most promising and recent non-invasive techniques is known under the acronym EVA, an ethylene vinyl acetate film which avoids contamination and proteins damages. This paper reports the results of the first-ever analysis of proteins extracted from the EVA film applied to a tissue sample some 40000 years old. In particular, the gut sample of a woolly mammoth (Mammuthus primigenus), discovered in 1972 close to the Shandrin River (Yakutia, Russia), via a shotgun MS-based approach was investigated. Proteomic and peptidomic analysis allowed the exploration of the mammoth dietary plants and its gut bacteria. The results were validated through the level of deamidation and other post-translational modifications of the peptides of the samples, which were used to discrimitate the “original” endogenous peptides from contaminant ones. Overall, the results of the metaproteomic analysis here reported are in accordance with the previous paleobotanical studies and with the reconstructed habitat of the Shandrin mammoth.