The antibiotic resistance (ARE) subfamily of ABC (ATP-binding cassette) proteins confers resistance to a variety of clinically important ribosome-targeting antibiotics and plays an important role in infections caused by pathogenic bacteria. However, inhibitors of ARE proteins have rarely been reported. Here, OptrA, a new member of the ARE proteins, was used to study inhibitors of these types of proteins. We first confirmed that destroying the catalytic activity of OptrA could restore the sensitivity of host cells to antibiotics. Then, fragment-based screening, a drug screening method, was used to screen for inhibitors of OptrA. The competitive saturation transfer difference experiments, docking, and molecular dynamics were used to determine the binding sites and mode of interactions between OptrA and fragment screening hits. In this study, we first find a novel and specific inhibitor of OptrA (CP1), which suppressed the ATPase activity of OptrA in vitro by 30%. A hydrogen bond formed between the 8-position phenylcyclic cyano group in CP1 and the amino acid residue Lys-271 allows CP1 to form a stable complex with OptrA protein. These findings provide a theoretical basis for the further optimization of the inhibitor structure to obtain inhibitors with higher efficiencies.