Pleurotus florida (Mont.) Singer is a mushroom species known to be an antioxidant, immunomodulatory, and diuretic agent, reducing blood pressure and cholesterol. The aim of this study was to evaluate the in vivo potency of P. florida's anti-diabetic properties in rats affected by hyperglycemia induced by Streptozotocin (STZ) at 55 mg/kg (i.p.), characterized by oxidative stress impairment, and changes in insulin levels and lipid profile. After inducing hyperglycemia in the rats, they were treated with P. florida acetone and methanol extracts, orally administered for 28 days at doses of 200 mg/kg and 400 mg/kg body weight. The hyperglycemic control (DC) group showed significant increases (P < 0.05) in mean blood sugar, total cholesterol, triglycerides, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, blood urea nitrogen, lipid hydroperoxides, and malondialdehyde, compared to the normal control (NC) group The high-density lipoprotein cholesterol, serum insulin, superoxide dismutase, catalase, glutathione disulfide, glutathione peroxidase, reduced glutathione, guaiacol peroxidase, and vitamin E and C levels showed a significant decrease (P < 0.05) in DC group, compared to the NC group. Blood glucose levels, lipid profiles, and insulin levels improved significantly after 28 days of treatment, in the group treated with glibenclamide (an oral hypoglycemic drug, used as positive control), and in the groups treated with P. florida extracts. In DC group, the treatment with P. florida was found to prevent diabetes, according to histopathological studies of the kidneys, pancreas, and liver of rats. In conclusion, this study has shown that the treatment with P. florida decreased oxidative stress and glucose levels in the blood, as well as restoring changes in lipid profiles.