Due to the lack of effective pharmacotherapy options to treats Alzheimer's disease, new strategies have been approached in the search for multi-target molecules as therapeutic options. In this work, four indole alkaloids, geissoschizoline, geissoschizone, geissospermine, and 3′,4′,5′,6′-tetradehydrogeissospermine were isolated from Geissospermum vellosii (Pao pereira) and evaluated for their anticholinesterase activities. While geissospermine inhibited only butyrylcholinesterase (BChE), the other alkaloids behaved as non-selective inhibitors of acetylcholinesterase (AChE) and BChE. In cell viability tests, only geissoschizoline was not cytotoxic. Therefore, geissoschizoline actions were also evaluated in human cholinesterases, where it was twice as potent inhibitor of hBChE (IC50 = 10.21 ± 0.01 µM) than hAChE (IC50 = 20.40 ± 0.93 µM). On enzyme kinetic studies, geissoschizoline presented a mixed-type inhibition mechanism for both enzymes. Molecular docking studies pointed interactions of geissoschizoline with active site and peripheral anionic site of hAChE and hBChE, indicating a dual site inhibitor profile. Moreover, geissoschizoline also played a promising anti-inflammatory role, reducing microglial release of NO and TNF-α at a concentration (1 μM) ten and twenty times lower than the IC50 values of hBChE and hAChE inhibition, respectively. These actions give geissoschizoline a strong neuroprotective character. In addition, the ability to inhibit hAChE and hBChE, with approximate inhibitory potencies, accredits this alkaloid for therapeutic use in the moderate to severe phase of AD. Thus, geissoschizoline emerges as a possible multi-target prototype that can be very useful in preventing neurodegeneration and restore neurotransmission. © 2020