| Summary: | This study aimed to characterize the chemical profile of an ethanolic extract of Tuscan <i>Rosmarinus officinalis</i> (<i>Ro</i>ex) and to determine its in vitro bioactivity. The content of phenolic and flavonoid compounds, hydroxycinnamic acids and triterpenoids was determined, and high-performance liquid chromatography-diode array detection (HPLC-DAD) analysis revealed that rosmarinic acid and other hydroxycinnamic derivatives were the main constituents of the extract. <i>Ro</i>ex demonstrated to have both antioxidant activity and the capability to scavenge hydrogen peroxide in a concentration dependent manner. Moreover, NIH3T3 mouse fibroblasts and human breast adenocarcinoma cells MDA-MB-231 viability was influenced by the extract with an IC<sub>50</sub> of 2.4 × 10<sup>−1</sup> mg/mL and 4.8 × 10<sup>−1</sup> mg/mL, respectively. The addition of <i>Ro</i>ex to the culture medium of both the above cell lines, resulted also in the reduction of cell death after H<sub>2</sub>O<sub>2</sub> pre-treatment. The Ames test demonstrated that <i>Ro</i>ex was not genotoxic towards both TA98 and TA100 strains, with and without S9 metabolic activation. The extract, by inactivating thrombin, showed to also have an anti-coagulating effect at low concentration values. All these biological activities exerted by <i>Ro</i>ex are tightly correlated to its phytochemical profile, rich in bioactive compounds.
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