Alzheimer disease and related dementias are major challenges, demanding urgent needs for earliest possible diagnosis to optimize the success rate in finding effective therapeutic interventions. Mounting solid scientific premises point at the core acetylcholine-biosynthesizing cholinergic enzyme, ChAT as a legitimate in vivo target for developing positron emission tomography biomarker for early diagnosis and/or monitoring therapeutic responses in the neurodegenerative dementias. Up-to-date, no PET tracer ligands for ChAT are available. Here we report for the first time a novel hierarchical virtual screening approach on a commercial library of ~300,000 compounds, followed by in vitro screening of the hits by a new High-Throughput ChAT assay. We report detailed pharmacodynamic data for three identified selective novel ChAT ligands with IC₅₀ and K_i values ranging from ~7 to 26 µM. In addition, several novel selective inhibitors of the acetylcholine-degrading enzymes, AChE and BuChE were identified, with one of the compounds showing an IC₅₀-value of ~6 µM for AChE. In conclusion, this report provides an excellent starting platform for designing and optimizing potent and selective ChAT ligands, with high potential as PET-imaging probe for early diagnosis of AD, and related dementias, such as Down’s syndrome and Lewy body disorders.