The leaves of Catharanthus roseus constitute the only source of the well known indolomonoterpenic alkaloids vincristine and vinblastine. In this work we studied the biological potential of the roots, which are used in several countries as decocts or hot water extracts for the treatment of a number of conditions. The aqueous extract strongly inhibited acetylcholinesterase (AchE) in an in vitro microassay, an effect ascribable mainly to serpentine (IC50 = 0.775 µM vs physostigmine IC50 = 6.45 µM) as assessed with the pure compound. Pure alkaloids were tested for muscarinic and nicotinic antagonism using rat ex-vivo preparations, namely, ileum and diaphragm/phrenic-nerve, respectively. Serpentine competitively blocked muscarinic receptors with a pA2 of 5.2, whereas the precursor ajmalicine up to 80 µM was undistinguishable from control, and catharanthine exhibited an unsurmountable muscarinic antagonism at greater than 10 µM concentrations. Nicotinic receptor mediated diaphragm contractions were fully inhibited by catharanthine (IC50 = 59.6 µM) and ajmalicine (IC50 = 72.3 µM), in a reversible but non-competitive manner, unlike the more potent nicotinic antagonist tubocurarine (IC50 = 0.35 µM) whose competitive blockade was overcome by a physostigmine-induced increase in acetylcholine. Serpentine up 100 µM did not change diaphragm contractions suggesting reduced affinity for neuromuscular nicotinic receptors. Despite strong in vitro AchE inhibition, serpentine failed to restore diaphragm contractions upon submaximal tubocurarine blockade, suggesting that poor tissue penetration may prevent serpentine from inhibiting AchE in deep neuromuscular synapses in the ex-vivo preparation. 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 the currently overlooked C. roseus roots may constitute a promising source of compounds with pharmaceutical interest. Moreover, given serpentine’s potent in vitro AchE inhibitory activity and low cholinergic receptor affinity, it is conceivable that minor structural modifications may yield a potent and selective AchE inhibitor, potentially useful for the pharmacological management of conditions such as Alzheimer’s disease and/or myasthenia gravis.