Inhibitors of acetylcholinesterase (AChE) are used in the treatment of Alzheimer's disease. Initial screening revealed that the edible flowers of N. pubescens inhibited AChE. This finding motivated us to identify the contributory bioactive metabolite(s) responsible for inhibition AChE using Gas Chromatography - Mass Spectrometry based metabolomics and chemometric approach. The metabolite levels as well as AChE inhibitory activity varied in different flower samples collected from different districts of West Bengal in the monsoon season. Different multivariate analyses differentiated the extracts with low activity (< 45% inhibition) from that of higher activities (> 45% inhibition). Chemometric analysis revealed gallic acid, a previously reported metabolite, to be one of the contributors significantly related to the inhibition of AChE by the flower extracts. Kaempferol, although negatively correlated, inhibited AChE. A probable synergistic action of metabolites is suggested for the AChE inhibitory property of N. pubescens flower extracts. Practical applications: Nymphaea pubescens flower is edible in different countries. The flowers also have different medicinal properties. Acetylcholinesterase inhibitors are used in the treatment of Alzheimer's disease. As the flower extracts of this plant inhibited acetylcholinesterase, although the activity of the flower extracts varied with locality of collection. The level of metabolites also varied. A detailed study was made to identify the active constituents. Gallic acid and kaempferol were found to have high acetylcholinesterase inhibitory activities. The study suggested that consumption of this edible part of the plant may have beneficiary effect on memory function. © 2018 Wiley Periodicals, Inc.