Anaerobic choline metabolism by human gut microbiota to produce trimethylamine (TMA) has recently evolved as a potential therapeutic target because of its association with chronic kidney disease and increased cardiovascular risks. Limited examples of choline analogs have been reported as inhibitors of bacterial enzyme choline TMA-lyase (CutC), a key enzyme regulating choline anaerobic metabolism. We utilized a new workflow to discover CutC inhibitors based on focused screening of a diversified library of small molecules for intestinal metabolic stability followed by in vitro CutC inhibitory assay. This workflow identified a histidine-based scaffold, (compound 5), as a CutC inhibitor with an IC50 value of 1.9 ± 0.2 μM. Remarkably, compound 5 was able to reduce the production of TMA in whole cell assays using various bacterial strains as well as in complex gut microbiota environment. The improved efficiency of the new scaffold identified in this study in comparison to previously reported CutC inhibitors would enable optimization of potential leads for in vivo screening and clinical translation. Finally, docking studies and molecular dynamic simulations were used to predict putative interactions created between inhibitor and CutC.© 2020 Wiley-VCH GmbH.