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Proton pump inhibitors (PPI) may improve symptoms in functional dyspepsia (FD) through duodenal eosinophil-reducing effects. However, the contribution of the microbiome to FD symptoms and its interaction with PPI remains elusive. Aseptic duodenal brushings and biopsies were performed before and after PPI intake (4 weeks Pantoprazole 40 mg daily, FD-starters and controls) or withdrawal (2 months, FD-stoppers) for 16S-rRNA sequencing. Between- and within-group changes in genera or diversity and associations with symptoms or duodenal factors were analyzed. In total, 30 controls, 28 FD-starters and 19 FD-stoppers were followed. Mucus-associated <i>Porphyromonas</i> was lower in FD-starters vs. controls and correlated with symptoms in FD and duodenal eosinophils in both groups, while <i>Streptococcus</i> correlated with eosinophils in controls. Although clinical and eosinophil-reducing effects of PPI therapy were unrelated to microbiota changes in FD-starters, increased <i>Streptococcus</i> was associated with duodenal PPI effects in controls and remained higher despite withdrawal of long-term PPI therapy in FD-stoppers. Thus, duodenal microbiome analysis demonstrated differential mucus-associated genera, with a potential role of <i>Porphyromonas</i> in FD pathophysiology. While beneficial effects of short-term PPI therapy were not associated with microbial changes in FD-starters, increased <i>Streptococcus</i> and its association with PPIeffects in controls suggest a role for duodenal dysbiosis after long-term PPI therapy.