In recent years, agricultural waste materials and wild plants have become alternative raw materials for the source of bioactive components. This study included the data from antioxidant capacity (DPPH, ABTS, CUPRAC, FRAP, phosphomolybdenum, and metal chelating) and enzyme inhibitory assays (cholinesterase, tyrosinase, alpha-amylase, and alpha-glucosidase) on Gossypium hirsutum L. stalk extracts as well as HPLC technique. Flavonoid contents of the extracts were found to be low, while the amounts of phenolics were found as 14.38 and 13.22 A mu mol gallic acid equivalents (GAEs)/g dry plant (dp) in the methanolic and aqueous extracts, respectively. The extracts were determined to contain significant amounts of apigenin, quercetin, (-)-epicatechin, and protocatechuic acid. The extracts exhibited remarkable antioxidant activity almost in all tests. In addition, the methanolic and aqueous extracts showed promising inhibitory activity on alpha-glucosidase. Phenolics, in particular, p-hydroxybenzoic and benzoic acids, were found to be in correlation with the activities of the extracts. It was concluded that the stalk, which is the post-harvest field trash of the cotton cultivation, is an alternative source of bioactive molecules and can be used in pharmaceutical and food industries for its anti-diabetic and antioxidant activities.