The present study was designed to characterize the polyphenols isolated from Acacia mearnsii bark crude extract (B) and fractions (B1-B7) obtained by high-speed counter-current chromatography (HSCCC) and evaluate their anti-inflammatory and carbolytic enzymes (α-glucosidase and α-amylase) inhibitory activities. Fractions B4, B5, B6, B7 (total phenolics 850.3, 983.0, 843.9, and 572.5 mg·g −1, respectively; proanthocyanidins 75.7, 90.5, 95.0, and 44.8 mg·g −1, respectively) showed significant activities against reactive oxygen species (ROS), nitric oxide (NO) production, and expression of pro-inflammatory genes interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS) in a lipopolysaccharide (LPS)-stimulated mouse macrophage cell line RAW 264.7. All the extracts suppressed α-glucosidase and α-amylase activities, two primary enzymes responsible for carbohydrate digestion. A. mearnsii bark samples possessed significantly stronger inhibitory effects against α-glucosidase enzyme (IC 50 of 0.4–1.4 μg·mL −1) than the pharmaceutical acarbose (IC 50 141.8 μg·mL −1). B6 and B7 (IC 50 17.6 and 11.7 μg·mL −1, respectively) exhibited α-amylase inhibitory activity as efficacious as acarbose (IC 50 15.4 μg·mL −1). Moreover, B extract, at 25 μg·mL −1, significantly decreased the non-mitochondrial oxidative burst that is often associated with inflammatory response in human monocytic macrophages.