The tropane alkaloid anisodamine (2) is obtained by 6β-hydroxylation of hyoscyamine (1). The application of this alkaloid in medicine is gaining attention due to the wide range of therapeutic applications described in addition to its anticholinergic activity. In this work, the production of anisodamine (2) by in vitro cultures of Brugmansia candida (Argentinean and Colombian samples) was studied. This alkaloid was estimated in different organs of in vitro-germinated seedlings as well as in hairy roots obtained from seedlings from both sources. Colombian roots exhibited the highest content of tropane alkaloids, with anisodamine (2) being the main alkaloid measured. In the leaves, the main alkaloid was scopolamine (3) and no significant differences were observed between Argentinean and Colombian leaves. The tropane alkaloid content in Argentinean hairy roots was significantly higher than in Colombian ones. Also, in the Argentinean samples the main alkaloid detected was anisodamine (2). Argentinean and Colombian B. candida seedlings and hairy roots appear to be a promising system for the production of anisodamine (2).