The community of Colonna (Rome, Central Italy), dated back to 8th–10th century CE, was characterized by a poor quality of life. In the present archaeobotanical investigation, we obtained a detailed qualitative reconstruction of the food habits of this Medieval population. To do it, light microscopy and GC–MS techniques were applied on the dental calculus of specimens exhumed from the cemetery of Colonna archaeological site, an approach never performed before on Italian Medieval samples. The identification of starch grains and other plant microfossils (e.g. pollen, phytoliths) showed a nutritional plan coherent with coeval poor classes, consisting of both C3 (e.g. Triticum sp.) and C4 (e.g. Sorghum bicolor) plant species, with a particular intake of acorns (Fagaceae) and legumes (Fabaceae) rich in proteins. Chromatographic analysis revealed the use of a large variety of foods: oil-rich seeds and fruits, aquatic resources (e.g. fish, molluscs, algae), animal derivatives (e.g. eggs, meat, milk, dairy products), Brassicaceae, Rosaceae fruits, aromatic herbs (Lamiaceae), mushrooms, wine and tea. In addition, specific markers of medicinal plants (e.g. Digitalis sp., Hyssopus officinalis, Artemisia sp., Ephedra sp.) evidenced the pharmaceutical culture of this historical period. Finally, the present work also amplified the knowledge about past environment, trade, plant uses and customs.