Esomeprazole, a proton pump inhibitor (PPI), is commonly prescribed for gastric-acid-related disorders but has been associated with impaired gastrointestinal (GI) motility with long-term use. However, the effect of concurrent antibiotic administration on this dysfunction remains unclear. Therefore, this study aimed to investigate the effects of antibiotics on esomeprazole-induced GI motility dysfunction and explore the underlying mechanisms in a mouse model. Male C57BL/6 mice were orally administered esomeprazole (160 mg/kg) five times per week for 4 weeks. Three days before initiating esomeprazole treatment, a broad-spectrum antibiotic cocktail (ABX) consisting of ampicillin (1 g/kg), neomycin (1 g/kg), metronidazole (1 g/kg), and vancomycin (0.5 g/kg) was provided in drinking water and maintained throughout the experimental period. Mosapride (3 mg/kg), a prokinetic agent, was used as a positive control. Neither esomeprazole alone nor in combination with ABX affected body weight or food intake. Compared to normal controls, esomeprazole treatment significantly delayed both intestinal transit and gastric emptying. However, ABX co-administration further pronounced intestinal transit time and improved gastric motility. The potential mechanisms may involve interactions among gastric H/K-ATPase, CYP3A11, gastrointestinal hormones (secretin and motilin), and the gut microbiome. Long-term esomeprazole use can impair both gastric and intestinal motility, and ABX co-treatment further exacerbates intestinal transit delay while paradoxically enhancing gastric emptying. These findings highlight the critical role of the gut microbiota in esomeprazole-induced GI motility dysfunction and suggest that antibiotic use should be approached with caution, particularly when combined with PPI therapy.