Fermented Nutrient Broth (FNB) was found to be a potential source of antidiabetic drugs in the previous report. Thus, FNB was employed to isolate the active compounds in this study. Novel 5 a-glucosidase inhibitors (aGIs) were isolated from FNB and identified as: (1) Adenine, (2) 3-hydroxyproline, (3). Creatinine, (4) Nicotinic acid, and (5) Niacinamide. Notably, these inhibitors were found as new microbial aGIs, non-sugar-based moiety aGls. Of these inhibitors, compounds 2 and 4 showed activity against rat a-glucosidase equal to, or stronger than, that of acarbose, with the IC50 values of 100, 67, and 115 mu g/mL, respectively. These results contributed to enriching the current poor novel microbial aGIs, and also confirmed that FNB shows promising utilization for diabetes and obesity treatments or health foods due to their containing efficient mammalian aGIs.