Hoppea fastigiata, an annual medicinal herb belonging to the Gentianaceae, is mostly found in South Asian countries, and is used by local tribes for various brain-related ailments. The genus possesses a unique class of compounds, xanthones, which are known for their potential against Alzheimer's and Parkinson's diseases. Limited availability and the potential pharmacological significance of the plants has led to the establishment of in vitro cultures of H. fastigiata and study of its neuroprotective principles. In vitro plantlets were established from the apical meristem of the plant in Murashige and Skoog medium with a combination of the phytohormones 6-benzylaminopurine (1 mg/L) and kinetin (0.1 mg/L), which was found to be efficacious with a growth index of 0.9 ± 0.01 after 30 days. Four different solvent extracts of in vitro cultures were evaluated for acetylcholinesterase (AChE) and monoamine oxidase A and B (MAO-A and MAO-B) inhibitory activities, amongst which the ethanolic extract showed the lowest IC50 value in all the assays. Three major compounds were isolated from the ethanolic extract and structurally confirmed as 1,5,7-trihydroxy-3-methoxyxanthone (1), 1,5-dihydroxy-3,7-dimethoxyxanthone (2) and 1,3,5-trihydroxy-8-methoxyxanthone (3). Compound 3 showed the strongest AChE inhibitory activity with mixed-type inhibition. Compounds 1 and 2 also showed promising AChE inhibitory properties with mixed and non-competitive types of inhibition, respectively. Compounds 1 and 2 showed inhibition of MAO-A (mixed and competitive, respectively) and compounds 2 and 3 showed inhibition of MAO-B (competitive and mixed, respectively). Extracts and isolated compounds showed good antioxidant capacities. The ethanolic extract and compound 2 showed the strongest antioxidant activities among the other solvent extracts and compounds, respectively.