One promising target for the development of novel antibiotics is trans-translation, a bacterial pathway which functions to rescue nonstop ribosomes. In a high-throughput screen for inhibitors of the pathway, a class of small molecules known as quinolines was discovered. Following organic synthesis, many compounds of the class had broad-spectrum activity against bacterial pathogens. In vivo reporters and molecular target-probing showed the quinoline class to inhibit proteolysis. The results suggest a novel function for quinoline antimicrobials.