AimsThe aim of this study was to screen endophytic fungi isolated from Vinca rosea for their potential to produce acetylcholinesterase (AChE) inhibitors. Method and ResultsEndophytic fungi isolated from V.rosea (Catharanthus roseus), were screened for AChE inhibitor production using Ellman's method. Maximum inhibition against AChE (78%) was observed in an isolate VS-10, identified to be Alternaria alternata on morphological and molecular basis. The isolate also inhibited butyrylcholinesterase (73%). Significant increase (13 fold) was achieved after optimization of process parameters using one variable at time approach. The inhibitor was purified using chromatographic techniques. The structure elucidation of the inhibitor was carried out using spectroscopic techniques and was identified to be altenuene'. The purified inhibitor possessed antioxidant potential as revealed by dot blot assay. The insecticidal potential of purified inhibitor was evaluated by feeding Spodoptora litura on diet amended with inhibitor. It evinced significant larval mortality. ConclusionsEndophytic A.alternata can serve as a source of dual cholinesterase inhibitor altenuene' with significant antioxidant and insecticidal activity. This is the first report on acetylcholinestearse inhibitory activity of altenuene. Significance and Impact of the StudyAlternaria alternata has the potential to produce a dual ChE inhibitor with antioxidant activity useful in the treatment of neurodegenerative disorders and in agriculture as biocontrol agent.