Antrodia cinnamomea (AC), an edible Taiwanese mushroom, has been recognized as a valuable natural resource with vast biological and medicinal benefits. Recently, the hypoglycemic and anti-diabetic effects of AC were mentioned in several studies. However, no studies have investigated α-glucosidase inhibitors from AC fruiting bodies (ACFB) as they relate to type 2 diabetes (T2D) treatment. The purpose of this study was to gain evidence of potent α-glucosidase inhibitory effects, as well as isolate, identify and characterize the active compounds of ACFB. The MeOH extract of ACFB demonstrated potent α-glucosidase inhibitory activity, and possessed high pH stability (pH 2–11) and thermostable properties at 40–50 °C. Further purification led to the isolation of eight constituents from ACFB, identified as: 25 S-antcin K ( 1), 25 R-antcin K ( 2), dehydrosulphurenic acid ( 3), 25 S-antcin I ( 4), 25 S-antcin B ( 5), 25 R-antcin B ( 6), dehydroeburicoic acid ( 7) and eburicoic acid ( 8). Notably, the ACFB extract and its identified compounds, except 1, 4, and 6 demonstrated a greater effect (EC 50 = 0.025–0.21 mg/mL) than acarbose (EC 50 = 0.278 mg/mL). As such, these active compounds were determined to be new potent mushroom α-glucosidase inhibitors. These active compounds were also identified on the HPLC fingerprints of ACFB.