Background: Ocotea species are known to produce secondary metabolites with a range of biological activities. This study aimed to evaluate the in vitro acetylcholinesterase (AChE) inhibition and neurotoxicity activities of the Ocotea aciphylla leaves. Materials and Methods: The in vitro anticholinesterase effect of crude extracts of O. aciphylla was investigated by means of spectrophotometric microplate assay. The most active extract, aqueous extract (AQE), was fractionated using column chromatography with silica gel as stationary phase to furnish several fractions that were also evaluated for the anticholinesterase effect. The neurotoxicity activity of AQE and active fraction (F9) was investigated in rat adrenal medulla pheochromocytoma strain cultures by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. The chemical characterization of the most active fraction was performed through high-performance liquid chromatography coupled with mass spectrometry multistage (HPLC-MS/MS). Results: Ethanolic extract (EE) and AQE exhibited significant inhibitory effects of the activity of AChE, with inhibitory concentration (IC50) of 443.7 and 412.8 μg/mL, respectively. Among the fractions, F9 was more effective AChE inhibition with IC50of 286.2 μg/mL. In the neurocytotoxicity assays, only the F9, at the highest concentration (500 μg/mL), induced a significant reduction in cell viability. HPLC-MS/MS analysis of the active fraction enabled the characterization of the natural compounds, i.e., procyanidin B-type dimer, propelargonidin dimer, catechin, and methoxy-luteolin-deoxyhexose-hexose. Conclusion: The leaves of O. aciphylla showed in vitro anticholinesterase activity and low neurotoxicity, and these effects might be related to the presence of phenolic compounds.