The antimycobacterial activity of the ethanolic extract from Berberis hispanica root bark was evaluated against Mycobactérium smegmatis and M. aurum. The extract was fractionated and the bands responsible for the activity were identified by bioautography. The results revealed the existence of three of four bands responsible for the antimycobacterial activity of the plant. A phytochemical study evidenced the nature of the main classes of secondary metabolites in the crude extracts and in the active bands of the bioautography. The molecules shown to be responsible for the antimycobacterial activity of the plant included alkaloides, polyphenols and flavonoids.