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Herein, we report a novel whole-cell screening assay using <i>Lactobacillus casei</i> as a model microorganism to identify inhibitors of energy-coupling factor (ECF) transporters. This promising and underexplored target may have important pharmacological potential through modulation of vitamin homeostasis in bacteria and, importantly, it is absent in humans. The assay represents an alternative, cost-effective and fast solution to demonstrate the direct involvement of these membrane transporters in a native biological environment rather than using a low-throughput in vitro assay employing reconstituted proteins in a membrane bilayer system. Based on this new whole-cell screening approach, we demonstrated the optimization of a weak hit compound (<b>2</b>) into a small molecule (<b>3</b>) with improved in vitro and whole-cell activities. This study opens the possibility to quickly identify novel inhibitors of ECF transporters and optimize them based on structure–activity relationships.