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Gastroesophageal reflux disease (GERD) leads to the accumulation of bile-induced reactive oxygen species and oxidative stress in esophageal tissues, causing inflammation and DNA damage. The progression sequence from healthy esophagus to GERD and eventually cancer is associated with a microbiome shift. Lactobacillus species are commensal organisms known for their probiotic and antioxidant characteristics in the healthy esophagus. This prompted us to investigate how Lactobacilli survive in a bile-rich environment during GERD, and to identify their interaction with the bile-injured esophageal cells. To model human reflux conditions, we exposed three Lactobacillus species (L. acidophilus, L. plantarum, and L. fermentum) to bile. All species were tolerant to bile possibly enabling them to colonize the esophageal epithelium under GERD conditions. Next, we assessed the antioxidant potential of Lactobacilli and role in bile injury repair: we measured bile-induced DNA damage using the ROS marker 8-oxo guanine and COMET assay. Lactobacillus addition after bile injury accelerated repair of bile-induced DNA damage through recruitment of pH2AX/RAD51 and reduced NFκB-associated inflammation in esophageal cells. This study demonstrated anti-genotoxic and anti-inflammatory effects of Lactobacilli, making them of significant interest in the prevention of Barrett’s esophagus and esophageal adenocarcinoma in patients with GERD.