The marine sponges with their unique and wide range of biodiversity, producing unusual metabolites emerge as a good candidate for new therapeutic agents, including as acetylcholinesterase (AChE) inhibitor. The aims of this study are to evaluate the potency of fractions and isolated compound from Agelas nakamurai as AChE inhibitor, as well as to determine the structure of the isolated compound. The bioassay-guided isolation protocol was carried out in this study. The AChE inhibitory assay was carried out based on the modified Ellman’s method. The structure of the isolated compound was determined by NMR spectroscopy and mass spectrometry. A diterpene alkaloid was isolated from the active fraction of A. nakamurai. This compound exhibited a molecular ion at m/z 422.3280 [M]+ in mass spectrometry. The UV profile of the isolated compound showed a strong peak at 269 nm, and the specific rotation value is [α]20D +28,0 (MeOH, c 0.25). These data together with the1H and13C NMR spectroscopy data are similar to that reported for iso-agelasine C. The isolated iso-agelasine C gave moderate inhibition against AChE enzyme with an IC50 value of 30.68 ± 0.92 µg/mL, which was lower compared to the extract and fractions. The stronger AChE inhibitory activity in the extract and fractions is possibly due to the synergistic activity of compounds present in the extract and fractions of A. nakamurai. Iso-agelasine C could serve as a lead for the development of diterpene alkaloid as an AChE inhibitor. © 2022 Marmara University Press.