Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting 47 million people worldwide. While acetylcholinesterase (AChE) inhibitors such as donepezil and galantamine are leading drugs in the symptomatic treatment of AD, new AChE inhibitors continue to be explored for improved potency and selectivity. Herein, a molecular networking approach using high resolution (HR-MS) and tandem mass spectrometry (MS2) has been used for rapid chemical profiling of an extract of the medicinal plant Vincetoxicum funebre Boiss. & Kotschy (Apocynaceae family) that was active against AChE. A total of 44 compounds were identified by combining the MN with traditional natural product methods, including the isolation and identification of five known compounds (13, 41-44) and a novel C13-norisoprenoid (40). In addition, the potential inhibitory activity of all 44 compounds was evaluated against the AChE enzyme via molecular docking to provide further support to the proposed structures. The glycosylated flavonoid querciturone (31) exhibited the highest affinity with a docking score value of −13.43 kJ/mol. Another five compounds showed stronger docking scores against AChE than the clinically used donepezil including the most active isolated compound daucosterol (44), with a binding affinity of −10.11 kJ/mol towards AChE. These findings broaden our understanding of Vincetoxicum metabolites and highlight the potential of glycosylated flavonoids as AChE inhibitors. Communicated by Ramaswamy H. Sarma.