Alzheimer’s disease (AD) is the most common form of dementia, which is recorded as a global health issue. Natural acetylcholinesterase inhibitors (AChEIs) are considered a helpful therapy for the management of symptoms of patients with mild-to-moderate AD. This work aimed to investigate and characterizeEuonymus laxiflorusChamp. (ELC) as a natural source of AChEIs compounds via in vitro and virtual studies. The screening parts used, including the leaves, heartwood, and trunk bark of ELC, revealed that the trunk bark extract possessed the highest activity, phenolics and flavonoid content. The in vitro anti-Alzheimer activity of ELC trunk bark was notably reclaimed for the first time with comparable effect (IC50= 0.332 mg/mL) as that of a commercial AChEI, berberine chloride (IC50= 0.314 mg/mL). Among various solvents, methanol was the most suitable to extract ELC trunk bark with the highest activity. Twenty-one secondary metabolites (1–21) were identified from ELC trunk bark extract, based on GCMS and UHPLC analyses. Of these, 10 volatile compounds were identified from this herbal extract for the first time. One phenolic (11) and seven flavonoid compounds (15–21) were also newly found in this herbal extract. Of the identified compounds, chlorogenic acid (11), epigallocatechin gallate (12), epicatechin (13), apigetrin (18), and quercetin (20) were major compounds with a significant content of 395.8–2481.5 μg/g of dried extract. According to docking-based simulation, compounds (11–19, and21) demonstrated more effective inhibitory activity than berberine chloride, with good binding energy (DS values: −12.3 to −14.4 kcal/mol) and acceptable RMSD values (0.77–1.75 Å). In general, these identified compounds processed drug properties and were non-toxic for human use, based on Lipinski’s rule of five and ADMET analyses.