Berberis empetrifolia L. and Berberis darwinii H. are native mountain plants of Chile and Argentina, characterised by their benzylisoquinolic alkaloid content. This research aims to analyse benzylisoquinoline alkaloids and their antibacterial activity in root extracts of Berberis empetrifolia L. and Berberis darwinii H. A methanol extract was separated by column chromatography through a polarity gradient and alkaloids were characterised by HPLC ESI-MS/MS. Antibacterial activity against Gram-positive and Gram-negative was assessed. The results showed the presence of protoberberine and aporphine alkaloids in the total extract and chloroform-methanol (CM) fraction. The antibacterial activity reports Staphylococcus aureus and S. epidermidis inhibitions. The berberine content can contribute to strain inhibition due to the synergic effect with other metabolites. A varietal effect could explain the difference in results between the species evaluated. This research generates new visions in the study of endemic species in the search for bioactive compounds of pharmacological interest.