Background State-of-the-art renewal has indicated the improvement of diagnostics of patients with metabolic associated fatty liver disease (MAFLD) and/or type II diabetes mellitus (T2DM) by dissecting the clinical characteristics as well as genomic analysis. However, the deficiency of the characterization of microbial and metabolite signatures largely impedes the symptomatic treatment. Methods For the purpose, we retrospectively analyzed the clinical data of 20 patients with MAFLD (short for "M"), 20 cases with MAFLD and T2DM (short for "MD"), together with 19 healthy donors (short for "Ctr"). Microbial and metabolite analyses were further conducted to explore the similarities and differences among the aforementioned populations based on feces and blood samples, respectively. Results Compared with those in the Ctr group, patients with M or MD revealed multifaceted similarities (e.g., Age, ALP, LDL, BUN) and distinctions in clinical indicators of liver (e.g., BMI, ALT, PCHE, CAP). With the aid of microbial and metabolite analyses as well as bioinformatic analyses, we found that the characteristics of gut microbiota (e.g., abundance, hierarchical clustering, cladogram, species) and lipid metabolism (e.g., metabolite, correlation coefficient and scatter plot) were distinct among the indicated groups. Conclusions The patients with MD revealed multifaceted similarities and distinctions in characteristics of microbiome and metabolites with those in the M and HD groups, and in particular, the significantly expressed microbes (e.g., Elusimicrobiota, Berkelbacteria, Cyanobacteria, Peregrinibacteria) and lipid metabolites (e.g., Lipid-Q-P-0765, Lipid-Q-P-0216, Lipid-Q-P-0034, Lipid-Q-P-0800), which would collectively benefit the clinical diagnosis of MAFLD and T2DM.