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Background: Phenolic compounds (PC) are found in various foods that are part of the human diet, such as mushrooms. The identification of PC is crucial due to their bioactive properties and their role in disease prevention. This study aimed to assess the antioxidant and antidiabetic properties of the phenolic component extract (PCE) derived from the Pleurotus ostreatus mushroom. The extract was separated using high-performance liquid chromatography (HLPC). The PCE was then tested on diabetic rats fed a high-fat diet (HFD). Also, this study was an attempt to treat infertility caused by type 2 diabetes in male rats. Methods: Streptozotocin (STZ) was administered to Wistar rats after a 4-week HFD as a model of type 2 diabetes (D2). Animals were given phenols orally on a high-fat diet for four weeks after being diagnosed with diabetes. Results: PCE constituents were 48.02% carbohydrates, 21.15% protein, 6.16% fiber, 2.06% fat, and 6.93% ash. The evaluation of the PCE was carried out by assessing its retention rates and subsequently comparing the UV data to standards. Total phenols and flavonoids in the crude extract were 23.04 mg/g gallic acid equivalent and 8.14 mg/g quercetin equivalent, respectively. Catechin was found as the significant PC (121.53 µg/mL), while gallic acid was the second PC (11.64 µg/ml) in the PCE of mushrooms. PCE showed high radical scavenging activity on DPPH. With dosage and incubation duration in dark-dependent conditions, it was discovered to be 93.23% at a 500 µg/ml concentration after 120 min in the dark. PCE reduces blood glucose levels while elevating insulin levels and antioxidant activity indicators. Our results show that phenols and their bioactive components can change glucose metabolism in STZ-induced diabetic HFD rats (D2). Conclusions: This study supports PCE's therapeutic potential in treating hyperglycemia, increasing insulin secretion, and addressing diabetes-induced infertility. [ABSTRACT FROM AUTHOR]