Catharanthus roseus remains one of the most important medicinal plants worldwide. The leaves of this species are the only source of the indolomonoterpenic alkaloids vincristin and vinblastin, whose anticancer activity represents powerful therapeutics to diseases, such as Hodgkin lymphoma. Usually, the remaining plant parts go to waste. Here we describe a phytochemical and bioactivity study on this species roots. Alkaloids in aqueous extracts were studied using HPLC-DAD-ESI-MS/MS, and serpentine and its isomer were found to predominate (64.7%) over other alkaloids, namely vindolinine and its isomer (23.9%), catharanthine (7.7%) and ajmalicine (3.8%). The aqueous extract strongly inhibited acetylcholinesterase (AchE), in an in vitro microassay, and effect ascribable mainly to serpentine (IC50 = 0.784 µM vs physostigmine IC50 = 6.45 µM) as assessed with the purified compound. The purified alkaloids were tested for cholinergic, namely muscarinic, antagonism using the rat ileum preparation. Serpentine competitively blocked muscarinic receptors with a pA2 of 5.2 µM, whereas the precursor ajmalicine up to 80 µM was undistinguishable from control, and catharanthine exhibited an unsurmoutable muscarinic antagonism at greater than 10 µM concentrations. To our knowledge,the present study is the first to assess the effect of C. roseus rootextracts, as well as of serpentine, ajmalicine and catharanthine on AchE. The results described herein suggest that C. roseus roots may constitute a promising source of bioactive compounds for the treatment of diseases benefiting from AchE inhibition, e.g. myasthenia gravis or Alzheimer’s disease. Moreover, we propose serpentine as possible lead compound for the development of novel AchE inhibitors.