α-Glucosidase (AGS) inhibitors have been regarded as an ideal target for the management of type 2 diabetes mellitus (T2DM) since they can maintain an acceptable blood glucose level by delaying the digestion of carbohydrates and diminishing the absorption of monosaccharides. In the process of our endeavor in mining AGS inhibitors from natural sources, the culture broth of two mangrove-derived actinomycetesStreptomycessp. WHUA03267 andStreptomycessp. WHUA03072 exhibited an apparent inhibitory activity against AGS. A subsequent chemical investigation into the two extracts furnished 28 secondary metabolites that were identified by spectroscopic methods as two previously undescribed linear polyketides1–2, four benzenoid ansamycins3–6, fourteen cyclodipeptides7–18, one prenylated indole derivative19, two fusicoccane-type diterpenoids20–21, two hydroxamate siderophore22–23, and five others24–28. Among all of the isolates,11and24were obtained from actinomycetes for the first time, while20–21had never been reported to occur in a marine-derived microorganism previously. In the in vitro AGS inhibitory assay, compounds3,8,9,11,14,16, and17exhibited potent to moderate activity with IC50values ranging from 35.76 ± 0.40 to 164.5 ± 15.5 μM, as compared with acarbose (IC50= 422.3 ± 8.4 μM). The AGS inhibitory activity of3,9,14,16, and17was reported for the first time. In particular, autolytimycin (3) represented the first ansamycin derivative reported to possess the AGS inhibitory activity. Kinetics analysis and molecular docking were performed to determine the inhibition types and binding modes of these inhibitors, respectively. In the MTT assay,3,8,9,11,14,16, and17exhibited no apparent cytotoxicity to the human normal hepatocyte (LO2) cells, suggesting satisfactory safety of these AGS inhibitors.