Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as nonalcoholic fatty liver disease (NAFLD), is associated with altered gut microbiota; however, there has been a focus on fecal samples, which are not representative of the entire digestive tract. Mucosal biopsies of the descending duodenum were collected. Five regions of the 16S rRNA gene were amplified and sequenced. Other assessments conducted on the study subjects included body mass index, transient elastography, liver enzymes, and lipid profile. Fifty-one subjects (36 with MASLD and 15 controls) were evaluated. There was no significant difference between the two groups regarding alpha- or beta-diversity of the duodenal mucosal microbiota. Linear discriminant analysis effect size (LEfSe) analysis showed that the genera Serratia and Aggregatibacter were more abundant in the duodenal mucosa of patients with MASLD, whereas the duodenal mucosal microbiota of the healthy controls was enriched with the genus Petrobacter. PICRUSt2 analysis revealed that genes associated with amino acid degradation and carboxylate degradation were significantly enriched in the duodenal mucosal microbiota of patients with MASLD. Our findings reveal the duodenal mucosal microbiota in patients with MASLD, which could contribute to future studies investigating the causal relationship between duodenal microbiota and MASLD.