Due to increasing mupirocin resistance, alternatives for nasal decolonization are urgently needed. Adhesion inhibitors are promising new preventive agents that may be less prone to induce resistance, as they do not interfere with the viability of and therefore exert less selection pressure. We identified promising adhesion inhibitors by screening a library of 4208 compounds for their capacity to inhibit adhesion to A-549 epithelial cells in vitro in a novel automated, imaging-based assay. The assay quantified DAPI-stained nuclei of the host cell; attached bacteria were stained with an anti-teichoic acid antibody. The most promising candidate, aurintricarboxylic acid (ATA), was evaluated in a novel persistent nasal colonization model using a mouse-adapted strain. Colonized mice were treated intranasally over 7 days with ATA using a wide dose range (0.5-10%). Mupirocin completely eliminated the bacteria from the nose within three days of treatment. In contrast, even high concentrations of ATA failed to eradicate the bacteria. To conclude, our imaging-based assay and the persistent colonization model provide excellent tools to identify and validate new drug candidates against nasal colonization. However, our first tested candidate ATA failed to induce decolonization.