Berberis aetnensisC. Presl (Berberidaceae) is a bushy-spiny shrub common on Mount Etna (Sicily). We demonstrated that the alkaloid extract of roots ofB. aetnensisC. Presl contains prevalently berberine and berbamine, possesses antimicrobial properties, and was able to counteract the upregulation evoked by glutamate of tissue transglutaminase in primary rat astroglial cell cultures. Until now, there are no reports regarding antioxidant properties ofB. aetnensisC. Presl collected in Sicily. Air-dried, powdered roots ofB. aetnensisC. Presl were extracted, identified, and quantified by HPLC. We assessed in cellular free system its effect on superoxide anion, radicals scavenging activity of antioxidants against free radicals like the 1,1-diphenyl-2-picrylhydrazyl radical, and the inhibition of xanthine oxidase activity. In primary rat astroglial cell cultures, exposed to glutamate, we evaluated the effect of the extract on glutathione levels and on intracellular production of reactive oxygen species generated by glutamate. The alkaloid extract ofB. aetnensisC. Presl inhibited superoxide anion, restored to control values, the decrease of GSH levels, and the production of reactive oxygen species. Potent antioxidant activities of the alkaloid extract of roots ofB. aetnensisC. Presl may be one of the mechanisms by which the extract is effective against health disorders associated to oxidative stress.