Islands are globally recognized biodiversity hotspots but remain highly vulnerable to invasive species. Among these, the house mouse (Mus musculus) is a widespread invasive rodent, known to impact native island ecosystems significantly. While extensive research has been conducted on its diet and ecological effects in oceanic islands, data from the Mediterranean region are scarce. This study provides a first overview of the diet of house mice from three Italian islands (San Domino, Ventotene, and Pantelleria) using a DNA metabarcoding approach. By analysing gut content and faecal samples, we identified 172 Amplicon Sequence Variants (ASVs), including 78 invertebrate, 46 plant, and 3 vertebrate ASVs. Similarly to what emerged from previous studies on oceanic islands, invertebrate consumption was dominated by Lepidoptera, with Noctuidae and Nymphalidae as the most represented families, while plant consumption was primarily composed of Fabaceae and Poaceae. However, no evidence of predation on native vertebrates was found and the only vertebrate sequences found were attributable to human food, indicating possible human-mice interactions on inhabited islands. Ventotene, the only rat-free island at the time of sampling, exhibited the widest niche breadth, suggesting that the absence of mammalian competitors may influence mouse feeding behaviour. These findings highlight the need for a context-specific approach when assessing rodent impacts in the Mediterranean and confirm the possible negative effects of mice on arthropods island populations.