Avian pathogenic E. coli (APEC), an extra-intestinal pathogenic E. coli (ExPEC), causes high morbidity and mortality in chickens. A recent report has also suggested APEC as a foodborne human uropathogen. Currently, antibiotics are commonly used to control APEC infections; however, APEC strains resistant to multiple antibiotics have been reported worldwide. Therefore, new generation anti-APEC therapeutics are critically needed. Here, we measured the efficacy of eight anti-APEC small molecule growth inhibitors (GIs) identified earlier from our in vitro study. Three GIs (GI-7, GI-10, and GI-6), when administered orally (1 mg/kg body weight), reduced the mortality (65.71% to 71.42%), lesions (perihepatitis, pericarditis, airsacculitis, and lung congestion) severity (13% to 62%) and APEC load (1.4 to 2.6 logs) in chickens. Further, GI-7 treatment at optimized dose (60 mg/L) in drinking water, under field simulated conditions, also reduced the mortality (80%), lesions severity (up to 63%) and APEC load (up to 2.1 logs) in chickens, which is superior compared to currently used antibiotic sulfadimethoxine. Pharmacokinetic analysis revealed that GI-7 is rapidly absorbed (0.5 h) in chickens with residue accumulation below FDA permissible limits (<0.1 ppm) in muscle, liver and kidney. The expression of outer membrane lipopolysaccharide transporter complex (LptD/E) was downregulated with depleted level of LptE and likely formation of non-functional LptD intermediate in APEC treated with GI-7. These results indicate that GI-7 can represent a novel anti-APEC therapeutic. Our future studies will be focused on developing the formulations to enhance the efficacy of GI-7 as well advancing GI-7 into field applications.