Pleurotus ostreatus is a macro-mushroom known worldwide for its taste, high nutritional value, and medicinal properties, and was one of the first cultivated mushroom. In this study, the extraction conditions of mushroom powders of mature fruit body (OF) and overripe fruit body (AOF) were optimized by Response Surface Method (RSM). Besides, the total phenolic content (TPC), antioxidant activity (DPPH, FRAP, and ABTS), and the inhibition effect of alpha-amylase and alpha-glucosidase, which are key enzymes in hyperglycemia, have been demonstrated for mushroom powders extracted under optimum conditions. TPC was lost 6.93 %, DPPH, FRAP and ABTS values 19.48 %, 24.87 %, and 16.91 % respectively in over-ripe fruits. In addition, alpha-glucosidase inhibition potential decreased by 7.33 % with delay of harvest time. In order to exhibit higher bioactive properties of P. ostreatus, flush time should be considered.