Background and aim: The prevalence and adverse outcomes of metabolic dysfunction associated with steatotic liver disease (MAFLD) are increasing. The changes in the gut microbiota and metabolites associated with metabolic dysfunction-associated steatohepatitis (MASH) are regarded as an essential part of the progression of MAFLD. This study aimed to identify the gut microbiota and metabolites involved in the development of MAFLD in patients. Method: This study enrolled 90 patients (healthy controls, HC: n = 30; MASH: n = 30; MASH-related cirrhosis, MC: n = 30), and their fecal samples were collected for 16S rRNA sequencing and non-targeted LC–MS/MS metabolomics analysis. Data preprocessing and statistical analyses were performed using QIIME2 software, Pynast, QIIME2 package, Progenesis QI, and R program. Results: The abundance of Prevotellaceae at the family level and Prevotella at the genus level was lower in the MASH and NC samples than in the HC samples. Both Prevotellaceae and Prevotella showed the strongest correlation with MASH progression via random forest analysis. Untargeted metabolomics was used to quantitatively screen for discrepant metabolites in the stool samples from the three groups. Linolenic acid (LA)-related metabolite levels were significantly lower in MASH and NC samples. Associations between Prevotella- or LA-related metabolites and liver function were discovered. A high abundance of Prevotella was associated with LA-related metabolites and MASH. Conclusion: This study identified that gut microbiota and metabolites are associated with MASH-related metabolic dysfunction. LA and Prevotella are depleted during MASH progression, and additional supplementation with Prevotella may be a potential strategy for the future treatment of MAFLD.