Non-alcoholic fatty liver disease (NAFLD) is closely associated with type 2 diabetes mellitus (T2D), and these two metabolic diseases demonstrate bidirectional influences. The identification of microbiome profiles that are specific to liver injury or impaired glucose metabolism may assist understanding of the role of the gut microbiota in the relationship between NAFLD and T2D. Here, we studied a biopsy-proven Asian NAFLD cohort (n = 329; 187 participants with NAFLD, 101 with NAFLD and T2D, and 41 with neither) and identified Enterobacter, Romboutsia, and Clostridium sensu stricto as the principal taxa associated with the severity of NAFLD and T2D, whereas Ruminococcus and Megamonas were specific to NAFLD. In particular, the taxa that were associated with both severe liver pathology and T2D were also significantly associated with markers of diabetes, such as fasting blood glucose and Hb1Ac. Enterotype analysis demonstrated that participants with NAFLD had a significantly higher proportion of Bacteroides and a lower proportion of Ruminococcus than a Korean healthy twin cohort (n = 756). However, T2D could not be clearly distinguished from NAFLD. Analysis of an independent T2D cohort (n = 185) permitted us to validate the T2D-specific bacterial signature identified in the NAFLD cohort. Functional inference analysis revealed that endotoxin biosynthesis pathways were significantly enriched in participants with NAFLD and T2D, compared with those with NAFLD alone. These findings may assist with the development of effective therapeutic approaches for metabolic diseases that are associated with specific bacterial signatures.