Juniperus brevifolia (Seub.) Antoine (Cupressaceae) is an endemic conifer of the Azores, a habitat isolated from the continental masses which may significantly influence the biosynthesis of bioactive compounds with unique features. In this context, the toxicity, antibacterial, antioxidant (DPPH) and anti-acetylcholinesterasic (anti-AChE) activities of the methanol and acetone extracts of J. brevifolia wood and bark were evaluated for the first time. In the brine-shrimp assay all the extracts showed low toxicity, with the concentration causing 50% mortality (LC 50 ) higher than 2.03 mg/mL. The acetone extract of the bark proved to be the most active against Bacillus cereus, B. subtilis and Micrococcus luteus, while the wood acetone extract showed activity only against B. cereus. No activity was observed against Gram (-) bacteria Escherichia coli and Enterobacter cloacae. The acetone extract of bark was the most active showing the higher antioxidant (EC 50 =7.0 µg/mL) and antiAChE activities (IC 50 =193 µg/mL), near to the reference compounds, quercetin (EC 50 =3.2 µg/mL) and ursolic acid (IC 50 =190 µg/mL), respectively. The present study demonstrates the great potential of the acetone bark extract of J. brevifolia as a source of new non-toxic natural bioactive compounds as antioxidant and antiacetylcholinesterasic drugs.