This study aimed to identify the gut microbiota features and metabolites associated with non-alcoholic fatty liver disease (NAFLD) among middle-aged and older adults. Using data from the Guangzhou Nutrition and Health Study (GNHS), a machine learning algorithm (lightGBM) was applied to analyze 272 metadata points from 1546 individuals. The top 12 taxa-related features of the microbiota were selected, and an individual microbiome risk score (MRS) was constructed. The MRS was positively associated with NAFLD, with an odds ratio (OR) of 1.86 (1.72, 2.02) per 1-unit increase in MRS. Specific gut microbiota, such as *f__veillonellaceae*, were associated with increased NAFLD risk, while *f__rikenellaceae*, *f__barnesiellaceae*, and *s__adolescentis* were associated with a decreased presence of NAFLD. Higher levels of bile acids, particularly taurocholic acid, were positively correlated with both a higher MRS and NAFLD risk. An animal fecal microbiota transplantation (FMT) experiment further confirmed the causal relationship between a higher MRS and NAFLD development. The study concluded that alterations in the core gut microbiota composition are biologically relevant to NAFLD development, highlighting the role of the microbiota in NAFLD pathogenesis.This study aimed to identify the gut microbiota features and metabolites associated with non-alcoholic fatty liver disease (NAFLD) among middle-aged and older adults. Using data from the Guangzhou Nutrition and Health Study (GNHS), a machine learning algorithm (lightGBM) was applied to analyze 272 metadata points from 1546 individuals. The top 12 taxa-related features of the microbiota were selected, and an individual microbiome risk score (MRS) was constructed. The MRS was positively associated with NAFLD, with an odds ratio (OR) of 1.86 (1.72, 2.02) per 1-unit increase in MRS. Specific gut microbiota, such as *f__veillonellaceae*, were associated with increased NAFLD risk, while *f__rikenellaceae*, *f__barnesiellaceae*, and *s__adolescentis* were associated with a decreased presence of NAFLD. Higher levels of bile acids, particularly taurocholic acid, were positively correlated with both a higher MRS and NAFLD risk. An animal fecal microbiota transplantation (FMT) experiment further confirmed the causal relationship between a higher MRS and NAFLD development. The study concluded that alterations in the core gut microbiota composition are biologically relevant to NAFLD development, highlighting the role of the microbiota in NAFLD pathogenesis.