Mamba intestinal toxin (MIT) is a minor toxin component of Dendroaspis polylepis venom and has been shown to have potent effects on intestinal smooth muscle. Since the publication of these findings more than 25 years ago, there has been little further investigation into the toxin. We used chromatography to fractionate the venoms of all Dendroaspis species and identified fractions containing MIT in D. polylepis and D. angusticeps venoms, both of which were confirmed by mass spectrometry. We next profiled the activity of purified MITs using in vitro assays relevant to the neurotoxic effects of these venoms. Both DaMIT and DpMIT showed moderate inhibition of the human muscle-type nicotinic acetylcholine receptor (nAChR) and weak binding to a chimera of the human alpha-7 nAChR and acetylcholine-binding protein. No other venom fractions from D. angusticeps showed inhibitory activity on the human muscle-type nAChR, which suggests that MIT may be responsible for the post-synaptic neurotoxicity previously observed for this venom. DaMIT and DpMIT both demonstrated moderate inhibitory activity of human voltage-gated potassium channels, and surprisingly, both were capable of fully neutralising the enzymatic activity of human acetylcholinesterase. Our work demonstrates that MIT may target several proteins at the neuromuscular junction to contribute to the neurotoxic effects of D. polylepis and D. angusticeps venoms.