In the course of a study of medicinal plants from Mali, the root bark of Zanthoxylum zanthoxyloides Lam. (Rutaceae) was investigated. The root bark of this plant is used as a toothbrush by West African people. Phytochemical investigation of the MeOH extract of Zanthoxylum zanthoxyloides Lam. (Rutaceae) led to the isolation of different biologically active compounds. The separation of acetylcholinesterase inhibitors was performed by centrifugal partition chromatography (CPC). The fractions were monitored by direct TLC bioautographic assays. The bio-guided isolation led to the isolation of a new peroxide derivative. In addition, LC/UV/MS analysis performed on the crude MeOH extract allowed on-line identification of some known compounds. The structures of the isolated compounds were elucidated by classical spectroscopic methods including UV, NMR, MS and HR-MS.