Background <p>Achalasia (AC) is clinically characterized by chronic intraluminal food stasis and bacterial fermentation, which may contribute to esophageal dysbiosis. This research seeks to analyze the microbiome of individuals with AC, pinpoint significant biomarkers, and explore their relationships with clinical and electrophysiological factors to enhance our comprehension of the mechanisms underlying AC.</p> Methods <p>This study, conducted at a single center, involved 16S rRNA sequencing of esophageal mucosal samples collected from individuals with achalasia and a control group. We assessed microbial diversity, identified distinct taxa using LEfSe, predicted functional capabilities through PICRUSt, and examined the relationship between microbiota and clinical parameters. Biomarkers were identified using a random forest algorithm and subsequently validated through qPCR.</p> Results <p>Patients with achalasia displayed notable dysbiosis in their esophageal microbiome, marked by changes in community structure while showing no significant shifts in diversity. There was a marked increase in the opportunistic bacterium <i>Fusobacterium</i>, alongside a reduction in beneficial species such as <i>Akkermansia</i> and <i>Ligilactobacillus</i>. The presence of <i>Pseudomonas</i> and <i>Ralstonia</i> was linked to the Eckardt score. The rise in <i>Fusobacterium</i> was further validated through qPCR analysis. Functional predictions suggested an increase in acetyl-CoA biosynthesis among these patients. Notably, levels of <i>Fusobacterium</i> were positively associated with critical clinical indicators, including pressures in the lower and upper esophageal sphincters and the Eckardt score.</p> Conclusions <p>Esophageal dysbiosis is associated with esophageal hypomotility in achalasia patients.</p>