This work aims to increase the floristic knowledge of southern Tuscany by means of a floristic investigation which took place in the Miniera di Murlo (southern Tuscany, Italy). The study area, located in the province of Siena, has an extension of 2.26 km² and hosts a heterogeneity of habitats like rocky outcrops, woodlands, meadows and riparian formations. The prevalent geological type is an acidic sedimentary rock known as diaspri formation (jasper). The checklist of vascular flora consists of 501 taxa and includes six endemics and 13 alien species, among which Vitislabrusca is a newly-found species for the Province of Siena along with the native Rosabalsamica and Rosasquarrosa. The most interesting species, such as Anthoxanthumaristatum, Gageabohemica, Paragymnopterismarantae, Saxifragagranulata, and Teesdaliacoronopifolia, are found on acidic rocks. Species of the Italian Red List, such as Ruscusaculeatus, and species of Regional conservation interest, such as Centaureaaplolepasubsp.carueliana, Ervilialoiseleurii, and Vicianigricans, were recorded. The chorological spectrum reveals a dominance of Mediterranean species indicating warm climate conditions and mild winters whereas the life-form spectrum shows a slight dominance of hemicryptophytes followed by therophytes. However, a clear dominance of one life-form type over the other is lacking, since the acidic and dry rocky substrate partially compensates for the humidity provided by the stream. This study highlights the relevant floristic value of the Miniera di Murlo area and supports the possible creation of a protected area or its inclusion in the adjacent one.