DNA barcode technique has only recently been applied to archaeobotanical studies. In fact, in association with morphological, scanning electron and optical microscopic analyses, these specific methods allow researcher to scientifically classify antique flora samples. Therefore, this project wants to improve, to encourage and spread further use of this protocol and to highlight the potentialities of the molecular biology and microscopy related to botanical fossils. In conclusion, ancient Olea europaea L. and Crataegus monogyna Jacq. seeds, a Pinus sp. pollen cone, a Quercus petraea (Mattuschka) Liebl. acorn, animal fibers and gymnosperm woody fragments, found in a 1st Century BC sunken Dressel 1B amphora, have clearly been identified, in order to enhance knowledge about Central Italy past human activity and environment. This research has also demonstrated the applicability of this scientific approach on specimens derived from underwater archaeological site.