Non-erosive reflux disease (NERD) pathogenesis has not been thoroughly evaluated. Here, we assessed the response of patients with NERD to proton pump inhibitor (PPI) therapy; changes in the microbiome and biologic marker expression in the esophageal mucosa were also evaluated. Patients with NERD (n = 55) received esomeprazole (20 mg) for eight weeks. The treatment response was evaluated at baseline, week four, and week eight. Esophageal mucosal markers and oropharyngeal and esophageal microbiomes were analyzed in patients who underwent upper gastrointestinal endoscopy at screening (n = 18). Complete and partial response rates at week eight were 60.0% and 32.7% for heartburn, and 61.8% and 29.1% for regurgitation, respectively. The expressions of several inflammatory cytokines, including IL-6, IL-8, and NF-κB, were decreased at week eight. Streptococcus, Haemophilus, Prevotella, Veillonella, Neisseria, and Granulicatella were prevalent regardless of the time-point (baseline vs. week eight) and organ (oropharynx vs. esophagus). The overall composition of oropharyngeal and esophageal microbiomes showed significant difference (P = 0.004), which disappeared after PPI therapy. In conclusion, half-dose PPI therapy for eight weeks could effectively control NERD symptoms. The expression of several inflammatory cytokines was reduced in the esophagus, and oropharyngeal and esophageal microbiomes in patients with NERD showed significant difference. However, the microbial compositions in the oropharynx and esophagus were not affected by PPI therapy in this study. Impact of PPI on the microbiome in patients with NERD should be more investigated in future studies.