Musa species are used traditionally for the management of many diseases. The study evaluated and compared anticholinesterase, anti‐inflammatory, antioxidant, and antidiabetic activities of Musa acuminata (Simili radjah, ABB) fruits and leaves fractions and characterized the bioactive compounds using HPTLC‐HRMS and NMR. Leaf fractions gave the higher biological activities than the fruit. Ethyl acetate fraction of the leaf had the highest total phenolic content (911.9 ± 1.7 mg GAE/g) and highest 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH·) scavenging activity (IC50, 9.0 ± 0.4 µg/ml). It also gave the most effective inhibition of acetylcholinesterase (IC50, 404.4 ± 8.0 µg/ml) and α‐glucosidase (IC50, 4.9 ± 1.6 µg/ml), but a moderate α‐amylase inhibition (IC50, 444.3 ± 4.0 µg/ml). The anti‐inflammatory activity of n‐butanol (IC50, 34.1 ± 2.6 µg/ml) and ethyl acetate fractions (IC50, 43.1 ± 11.3 µg/ml) of the leaf were higher than the positive control, quercetin (IC50, 54.8 ± 17.1 µg/ml). Kaempferol‐3‐O‐rutinoside and quercetin‐3‐O‐rutinoside (rutin) were identified as the bioactive compounds with antioxidant and antidiabetic activities from the ethyl acetate fraction of M. acuminata leaf. Practical applications All parts of Musa acuminata are known to be useful ethnomedicinally even as food. The leaves are mostly used to serve food and used for wrapping purposes. However, this study concluded that M. acuminata leaf is rich in bioactive flavonoids such as kaempferol‐3‐O‐rutinoside and rutin, with relatively high antioxidative, antidiabetic, and anti‐inflammatory activities. Therefore, aside the fact that the leaves can serve as potential drug leads for pharmaceutical industries, it can also be embraced in the food sector to produce supplements and/or nutraceuticals in the management of Alzheimer's, diabetes and other inflammatory diseases.